BRPI0709900A2 - Method and composition for inhibiting masp-2 dependent complement activation, and methods for manufacturing a medicament for treating an individual suffering from a masp-2 dependent complement mediated condition for treating and / or preventing atherosclerosis in an individual in need thereof to treat an individual suffering from malignancy, an endocrine disorder, and complement-mediated ophthalmic condition - Google Patents

Abstract

METHOD AND COMPOSITION FOR INHIBITING MASP-2 DEPENDENT COMPLEMENT ACTIVATION, AND METHODS FOR MANUFACTURING A MEDICINAL PRODUCT TO TREAT AN INDIVIDUAL SUFFERING FROM A CONDITION MEDIATED BY MASP-2 AND PRETENDING AERPOSE IN COMPLEMENT An individual in need thereof to treat an individual suffering from malady, an endocrine disorder and complement-mediated condition <D> In one aspect, the invention provides methods of inhibiting the effects of complement-dependent complement activation. -2 in a living individual. The methods comprise the step of administering to an individual in need thereof an amount of a MASP-2 inhibiting agent effective to inhibit MASP-2 dependent complement activation. In some embodiments, the MASP-2 inhibitory agent inhibits cell damage associated with activation of the MASP-2 mediated alternative complement pathway, leaving the classic (Clq-dependent) pathway component of the immune system intact. In another aspect, the invention provides compositions for inhibiting the effects of lectin-dependent complement activation comprising a therapeutically effective amount of a MASP-2 inhibiting agent and a pharmaceutically acceptable carrier.

Description

"METHOD AND COMPOSITION FOR INHIBITING DEPENDENT DEPENDENT ACTIVATION OF MASP-2, AND METHODS FOR MANUFACTURING A MEDICINAL PRODUCT TO TREAT A COMPLIANT MEDIATED CONDITION FOR MASP-2 DEPENDENT AND INCLUDING AND / OR INCLUDING SAME, TO TREAT AN INDIVIDUAL SUFFERING FROM A MALIGNITY, ENDOCRINE DISTURBANCE AND COMPLEMENT-MEDIATED OFTALMOLOGICAL CONDITION "

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation in part of US Application No. 2111 / 150,883, filed June 9, 2005, the priority of which is claimed under US 35§120, which claims benefit of US Provisional Application No. 60 / 578,847, filed June 10, 2005. June 2004, under 35U.SC§ 119 (e), and also claims the benefit of US Interim Application No. 60 / 788,876, filed April 3, 2006, under 35 USC§ 119 (e).

FIELD OF INVENTION

The present invention relates to methods of inhibiting the adverse effects of MASP-2 dependent complement activation.

BACKGROUND OF THE INVENTION

The complement system provides a mechanism of initial action to initiate and amplify the inflammatory response to microbial infection and other acute insults (K.. K. Liszewski and JP Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by WE Paul, RavenPress, Ltd., New York). Although complement activation provides valuable first-line defense against potential pathogens, complement-promoting activities that promote a protective inflammatory response may also pose a potential threat to the host (KR Kalli, etal., Springer Semin. Immunopathol. 15: 417-431, 1994; P. P. Morgan, Eur. J. Clinical Investig. 24: 219-228, 1994). For example, C3 and C5 proteolytic products recruit and activate neutrophils. These activated cells are discriminated in their release of destructive enzymes and can cause organ damage. In addition, complement activation may cause decomposition of lytic complement components near host cells as well as microbial targets, resulting in host cell lysis.

The complement system has been implicated as contributing to the pathogenesis of numerous acute and chronic disease states, including: myocardial infarction, revascularization following cerebrovascular accident, ARDS, reperfusion injury, septic shock, shedding following thermal burns, inflammation after cardiopulmonary bypass. , transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. In almost all of these conditions, complement is not the cause but is one of several factors involved in pathogenesis. Nevertheless, complement activation can be a major pathological mechanism and represents an effective point for clinical control in many of these disease states. The increasing recognition of the importance of complement-mediated tissue injury in a variety of disease states stresses the need for effective complement inhibitory drugs. No drug has been approved for human use that specifically targets and inhibits complement activation.

Currently, it is widely accepted that the decomposition system can be activated via three distinct pathways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is usually triggered by antibody bound to a foreign particle (i.e. an antigen) and thus requires prior exposure to this antigen for the generation of specific antibody. Since activation of the classical pathway is associated with the development of an immune response, the classical pathway is part of the acquired immune system. In contrast, both the lectin and alternative pathways are independent of clonal immunity and are part of the innate immune system.

The first step in activating the classical pathway is the binding of a specific recognition molecule, Clq, to antigen-linked IgG and IgM. Activation of the complement system results in the sequential activation of serine protease zymogens. Clq is associated with the serine protease enzymes Clr and Cls as a complex called Cl and, on binding of Clq to an immune complex, autoproteolytic cleavage of the ClrAr-IIe site is followed by Clr activation of Cls, which thus acquire the ability to cleave C4 and C2. Cleavage of C4 into two fragments, designated C4a and C4b, enables C4b fragments to form covalent bonds with adjacent hydroxyl or amino groups and subsequent generation of C3 convertase (C4b2b) through non-covalent interaction with activated C2 C2b deletion. C3 convertase (C4b2b) activates C3 leading to the generation of C5 convertase (C4b2b3b) and the formation of the membrane attack complex (C5b-9) that can cause microbial lysis. activated forms of C3 and C4 (C3b and C4b) are covalently deposited on the target foreign surfaces, which are recognized by multiple phagocyte complement receptors.

Independently, the first step in activating the breakthrough system by lectin pathway is also the binding of specific recognition molecules, which is followed by activation of serine proteases associated. However, instead of the binding of immune complexes by Clq, lectin pathway recognition molecules are proteins that bind carbohydrate (mannan-binding lectin (MBL), H-ficoline, M-ficoline and L-ficoline) (J. Lu et al Biochim, Biophys, Acta 1572: 387-400, 2002; Holmskov et al., Annu. Rev. Immunol. 21: 547-578 (2003); Teh et al., Immunology 101: 225-232 (2000)) . Ikeda et al. first demonstrated that, like Clq, MBL could activate the complement system in binding to yeast mannan-coated erythrocytes in a C4-dependent manner (K. Ikeda et al., J. Biol. Chem. 262: 7451-7454, 1987). MBL, a member of the collectin protein family, is a calcium-dependent lectin that binds carbohydrates with 3- and 4-hydroxy groups oriented in the equatorial plane of the pyranose ring. Prominent MBL ligands are thus D-mannose and N-acetyl-D-glycosamine, while carbohydrates that do not meet this steric requirement have undetectable affinity for MBL (Weis, WI, et al., Nature 360: 127-134, 1992). . The interaction between MBL and monovalent sugars is extremely weak, with dissociation constants typically within the 2 mM range. MBL achieves firm, specific binding to glycan ligands by interacting with monosaccharide residues multiple simultaneously (Lee, R. T., et al., Archiv. Biochem. Biophys. 299: 129-136,1992). MBL recognizes the carbohydrate patterns we usually coat with microorganisms such as bacteria, yeast, parasites and certain viruses. In contrast, MBL does not recognize D-galactose and sialic acid, the second to last sugars that usually coat "mature" complex glycoconjugates present in mammalian plasma and surface cell glycoproteins. This binding specificity is considered to help protect from self-activation. However, MBL does not bind with high affinity to the high mannose "precursor" glycan groups on N-linked glycoproteins sequestered in the endoplasmic reticulum and Golgi mammalian cells (Maynard, Y., et al., J. Biol. Chem. 257 : 3788-3794, 1982). Therefore, damaged cells are potential targets for pathway activation of lectin via MBL binding.

Phytolins have a different type of lectin domain than MBL, called the fibrinogen equivalent domain. Phytolines bind sugar residues in a manner independent of Ca "1" 1 ". In humans, three types of phytolines, L-ficoline, M-ficoline and H-ficoline, have been identified. have a specificity for N-acetyl-D-glycosamine, however H-phytoline also binds N-acetyl-D-galactosamine.The difference in sugar specificity of L-phytoline, H-phytoline and MBL means that different lectins may be complementary and target different, though overlapping, glycoconjugates.This concept is supported by the recent report that of the known lectins in the lectin pathway, only L-ficoline specifically binds to lipoteichoic acid, a cell wall glycoconjugate found in all Gram-positive bacteria. (Lynch, NJ, et al., J. Immunol. 172: 1198-1202, 2004). Collectins (ie MBL) and phycolines carry no similarity in amino acid sequence. However, the two groups Most of these proteins have similar domain organizations and, like Clq, aggregate into oligomeric structures that maximize the possibility of multiple site binding. Serum MBL concentrations are highly variable in healthy populations and this is genetically controlled by polymorphisms / mutations in both the promoter and coding regions of the MBL gene. As an acute phase protein, MBL expression is still over-regulated during inflammation. L-ficoline is present in serum at similar concentrations as MBL. Therefore, the L-ficoline branch of the lectin pathway is potentially comparable to the MBL branch in strength. MBL and phytolins can also function as opsonins, wanting the interaction of these proteins with phagocyte receptors (Kuhlman, M., et al. J. Exp. Med. 169: 1733 (1989; Matsushita, M., et al., J. Biol. Chem. 2771: 2448-54, 1996). However, the identities of the receptor (s) in phagocytic cells have not been established.

Human MBL forms a specific, high-affinity interaction through its collagen-equivalent domain with unique Clr / Cls-equivalent serinasproteases, called MBL-associated serine proteases (MASPs). So far, three MASPs have been described. First, a unique "MASP" enzyme has been identified and characterized as the enzyme responsible for the onset of the complement cascade (ie, cleaving C2 and C4) (Ji, YH, et al., J. Immunol. 150: 571-578, 1993 ). It was later confirmed that MASP is indeed a mixture of two proteases: MASP-I and MASP-2 (Thiel, S., et al., Nature 386: 506-510, 1997). However, it has been shown that MBL-MASP-2 complex alone is sufficient for complement activation (Vorup-Jensen, T., et al., J. Immunol. 165: 2093-2100, 2000). In addition, only MASP-2 cleaved C2 and C4 at high rates (Ambrus, G., etal., J. Immunol. 170: 1374-1382, 2003). Therefore, MASP-2 is responsible for activating C4 and C2 to generate C3 convertase, C4b2b. This is a significant difference from the Cl complex, where the coordinated action of specific duasserine proteases (Clr and Cls) leads to activation of the breakdown system. Recently, a third new protease, MASP-3, has been isolated (Dahl, M.R., et al., Immunity 15: 127-35, 2001). MASP-I and MASP-3 are alternatively joined products of the same gene. The biological functions of MASP-I and MASP-3 remain to be resolved.

MASPs share identical domain organizations with those of Clr and Cls, the enzymatic components of the Cl complex (Yes, R.B., et al., Biochem. Soc. Trans. 28: 545, 2000). These domains include an N-terminal Sea Urchin / Bone Morphogenic (CUB) Clr / Cls / Vegf protein domain, a domain equivalent to epidermal growth factor, a second CUB domain, a complement control protein tandem, and a serine protease domain. Like nasproteases Cl, MASP-2 activation occurs by cleavage of an Arg-Ile bond adjacent to the serine protease domain, which divides the enzyme into disulfide-linked AeB chains, the latter consisting of the deserine protease domain. Recently, a genetically determined MASP-2 deficiency has been described (Stengaard-Pedersen, K., et al., New Eng. J. Med.349: 554-560, 2003). Mutation of a single nucleotide leads to an Asp-Gly exchange in the CUB 1 domain and renders MASP-2 unable to bind to MBL.

MBL is also associated with a nonenzymatic protein alluded to as a 19 kDa MBL-associated protein (MApl9) (Stover, CM, J. Immunol. 162: 3481-90, 1999) or small MBL-associated protein (sMAP) (Takahashi, M (et al., Int. Immunol. 11: 859-863,1999). MAp 19 is formed by the alternate junction of the MASP 2e gene product comprising the first two domains of MASP-2, followed by an extra sequence of four unique amino acids. The MASP 1 and MASP 2 genes are located on chromosomes 3 and 1, respectively (Schwaeble, W., et al., Immunobiology 205: 455-466, 2002).

Several lines of evidence suggest that there are different MBL-MASPs complexes and a large fraction of total serum MASPs is not complexed with MBL (Thiel, S., et al., J. Immunol. 165: 878-887, 2000). Both H - L-ficoline is associated with MASP and activates the lectin complement pathway, as does MBL (Dahl, MR, et al., Immunity15: 127-35, 2001; Matsushita, M., et al., J (Immunol. 168: 3502-3506, 2002). Both the lectin and classical pathways form a common C3 (C4b2b) and the two paths converge at this stage.

The lectin pathway is widely considered to play a major role in defending the host against infection. Strong evidence for the development of MBL in host defense comes from the analysis of patients with decreased serum functional MBL levels (Kilpatrick, D. C., Biochim.Biophys. Acta 1572: 401-413, 2002). Such patients demonstrate susceptibility to recurrent bacterial and fungal infections. These symptoms are usually evident early in life, during a clear window of vulnerability as maternally derived antibody titers weaken, but before a full repertoire of anticorpose responses develops. This syndrome often results from mutations at various sites in the collagen portion of MBL, which interferes with the proper formation of MBL oligomers. However, since MBL may function as a complement-independent opsonin, it is not known to which degree the increased susceptibility to infection is due to impaired complement activation.

Although there is extensive evidence implicating both classical and alternative pathways in non-infectious human disease pathogenesis, the role of the lectin pathway is only just beginning to be evaluated. Recent studies provide evidence that lectin pathway activation may be responsible for complement activation and related inflammation in ischemia / reperfusion injury.Collard et al. (2000) reported that cultured endothelial cells subjected to oxidative stress bind MBL and show C3 deposition upon exposure to human serum (Collard, C. D., et al., Am. J. Pathol. 156: 1549-1556, 2000). In addition, treatment of human sera with blocking anti-MBL monoclonal antibodies inhibited MBL binding and complement activation. These findings were extended to a rat model of myocardial ischemia-reperfusion in which rats treated with a mouse MBL-directed anti-blocker showed significantly less myocardial injury in occlusion of a coronary artery than those treated with a control antibody (Jordan, JE, et al., Circulation 104: 1413-1418, 2001). The molecular mechanism of MBL binding to endotheliovascular after oxidative stress is uncertain; A recent study suggests that activation of the lectin pathway after oxidative stress may be mediated by the binding of MBL to vascular endothelial cytokeratins rather than glycoconjugates (Collard, CD, et al., Am. J. Pathol. 159: 1045-1054,2001 ). Other studies have implicated the classical and alternative pathways in the pathogenesis of ischemia / reperfusion injury and the role of the lectin pathway in this disease remains controversial (Riedermann, NC, et al., Am. J.Pathol. 162: 363-367, 2003). Unlike the classical and lectin pathways, no alternative pathway illuminator has been found to fulfill the recognizing functions that Clq and lectins perform in the other two pathways. It is widely accepted that the alternative pathway is spontaneously triggered by foreign surfaces or other abnormal surfaces (bacteria , yeast, virally infected or damaged tissue). There are four plasma proteins directly involved in the alternative pathway: C3, BeDe properdina factors. Proteolytic generation of C3b from native C3 is required for the alternate path to function. Since the C3b alternative pathway convertase (C3bBb) contains C3b as an essential subunit, the question as to the origin of the first C3b via the alternative pathway has been problematic and has stimulated considerable research.

C3 belongs to a family of proteins (together with C4 and α-2 macroglobulin) that contain a rare known post-translational modification as a thioester bond. The thioester group is made up of a glutaminacou which terminal carbonyl group is attached to the sulfhydryl group of a forward three amino acid cysteine. This bond is unstable and the carbonyl electrophilic group of glutamine may form a covalent bond with other molecules via hydroxyl or amino groups. The thioester bond is reasonably stable when sequestered within an intact C3 hydrophobic pouch. However, proteolytic cleavage of C3 to C3a and C3b will result in exposure of the highly reactive C3b thioester bond and by C3b stemecanism covalently binds to a target. In addition to its well-documented role in covalent binding of C3b to target complements, C3 thioester is also considered to play a central role in triggering the alternative pathway. According to the widely accepted "tick-over theory", the alternative pathway is initiated by the generation of a fluid phase convertase, iC3Bb, which is formed from C3 with hydrolyzed thioester (iC3; C3 (H20)) and factor B ( Lachmann, J., et al., Springer Semin (Immunopathol. 7: 143-162, 1984). C3b-iC3 equivalent is generated from native C3 by slow spontaneous hydrolysis of the internal thioester in the protein (Pangburn, M.K., et al., J.Exp. Med. 154: 856-867, 1981). Through the activity of iC3Bb convertase, C3b molecules are deposited on the target surface, thus starting the alternative pathway.

Very little is known about alternate path activation initiators. Activators are considered to include yeast (zymosan) cell walls, very pure polysaccharides, rabbit erythrocytes, certain immunoglobulins, viruses, fungi, bacteria, animal tumor cells, parasites, and damaged cells. The only common trait among these activators is the presence of carbohydrate, but the complexity and variety of carbohydrate structures has made it difficult to establish the shared molecular determinants that are recognized.

The alternative pathway can also provide a powerful amplification handle for the lectin / classical pathway C3 convertase (C4b2b) as any generated C3b can participate with factor B in the formation of the additional alternative pathway (C3bBb) C3 convertase. Alternate pathway C3convertase is stabilized by properdin binding.

Properdin prolongs the half-life of C3 convertase from the alternate path six to ten times. The addition of C3b to C3 convertase leads to the formation of the C5convertase of the alternate pathway.

All three pathways (that is, the classical, lectin and alternative) were considered to converge on C5, which is cleaved to form products with multiple proinflammatory effects. The converged path was changed as the terminal complement path. C5a is the most potent anaphylatoxin, inducing changes in smooth muscle and vascular tone, as well as vascular permeability. It is also a powerful chemotaxin with both neutrophil and monocyte activator. C5a mediated cellular activation can significantly amplify inflammatory responses by inducing release of multiple additional inflammatory mediators, including cytokines, hydrolytic enzymes, arachidonic acid metabolites, and reactive oxygen species. Cleavage of C5 leads to the formation of C5b-9, also known as the membrane attack complex (MAC). There is now strong evidence that sublimitic MAC deposition may play an important role in inflammation beyond its role as a lytic pore forming complex.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of inhibiting the adverse effects of MASP-2 dependent complement activation in a living patient. The method includes the step of administering to a patient in need thereof an amount of a MASP-2 inhibiting agent effective to inhibit MASP-2 dependent complement activation. In this context, the phrase "MASP-2 dependent complement activation" refers to alternative pathway activation that occurs through the lectin dependent MASP-2 system. In another aspect of the invention, the MASP-2 inhibitory agent inhibits complement activation via the lectin-dependent MASP-2 system without substantially inhibiting complement activation via the classical or Clq-dependent system, such that the Clq-dependent system remains functional. .

In some embodiments of these aspects of the invention, the MASP-2 inhibiting agent is an anti-MASP-2 or fragmented antibody. In other embodiments, the anti-MASP-2 antibody has reduced effector function. In some embodiments, the DEMASP-2 inhibiting agent is a MASP-2 inhibiting peptide or a non-peptide MASP-2 inhibitor.

In another aspect, the present invention provides compositions for inhibiting the adverse effects of MASP-2 dependent complement activation comprising a therapeutically effective amount of a MASP-2 inhibiting agent and a pharmaceutically acceptable carrier. Methods are also provided for the manufacture of a medicament for use in inhibiting the adverse effects of MASP-2-dependent complement activation in living patients in need, comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier. Methods are also provided for the manufacture of medicaments for use in inhibiting MASP-2 dependent complement activation for the treatment of each of the conditions, diseases, and disorders described hereinbelow.

The methods, compositions and medicaments of the invention are useful for inhibiting the adverse effects of MASP-2 dependent complement activation in vivo in mammalian patients, including humans who suffer from an acute or chronic pathological condition or injury as described herein. Such conditions and lesions include without limitation MASP-2-mediated activation activation in autoimmune disorders and / or associated inflammatory conditions.

In one aspect of the invention, methods are provided for treating ischemic reperfusion injury by treating a patient experiencing ischemic reperfusion, including without limitation, after repair of aortic aneurysm, cardiopulmonary bypass, vascular reanastomosis in connection with, for example, organ transplants (eg heart, lung, liver, kidney) and / or end-to-finger reimplantation, stroke, myocardial infarction, hemodynamic resuscitation following shock and / or surgical procedures, with a therapeutically effective amount of a MASP-2 inhibitor in a vehicle pharmacopharmaceutical.

In one aspect of the invention, methods are provided for inhibiting atherosclerosis by treating a patient suffering from or prone to atherosclerosis with a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier.

In one aspect of the invention, methods are provided for inhibiting MASP-2 dependent complement activation in a patient experiencing a vascular condition, including without limitation cardiovascular conditions, cerebrovascular conditions, vascular (peripheral), renovascular, mesenteric / vascular conditions. and revascularization for transplants and / or replants, such patient being treated with a therapeutically effective amount of a MASP-2 inhibiting agent. Such conditions include without limitation the treatment of: vasculitis, including Henoch-Schonlein purpura nephritis, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis (also called rheumatoid arthritis), immune complex vasculitis and Takayasu disease; dilated cardiomyopathy; diabetic angiopathy; Kawasaki disease (arteritis); gasvenous plunger (VGE); and / or restenosis following catheter placement, atherectomy, and / or percutaneous transluminal coronary angioplasty (PTCA).

In another aspect of the invention, methods are provided for inhibiting MASP-2-dependent complement activation in a patient suffering from inflammatory gastrointestinal disorders, including but not limited to pancreatitis, diverticulitis, and intestinal disorders including Crohn's disease, ulcerative colitis, and syndrome. irritable bowel.

In another aspect of the invention, methods are provided for inhibiting MASP-2-dependent complement activation in a patient suffering from a pulmonary condition including but not limited to acute respiratory distress syndrome, transfusion-related acute lung injury, acute ischemia / lung injury. reperfusion, chronic obstructive pulmonary disease, asthma, Wegener's granulomatosis, antiglomerular basement membrane disease (Goodpasture's disease), meconium aspiration syndrome, bronchiolitis obliterative syndrome, pulmonary pulmonary disease, burn-related acute pulmonary depression, cardiogenic pulmonary depression, related respiratory depression with transfusion and emphysema.

In another aspect of the invention, methods are provided for inhibiting MASP-2-dependent complement activation in a patient who has undergone, is undergoing or will undergo extracorporeal reperfusion procedure, including but not limited to hemodialysis, plasmapheresis, leukopheresis, membrane oxygenation. (ECMO), LDL precipitation on heparin-induced extracorporeal membrane oxygenation (HELP) and cardiopulmonary bypass (CPB).

In another aspect of the invention, methods are provided for inhibiting MASP-2-dependent complement activation in a patient suffering from a musculoskeletal condition, including but not limited to osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, gout, neuropathic arthropathy, psoriatic arthritis, ankylosing spondylitis or other crystalline arthropathies and response, muscular dystrophy or systemic lupuseritismatosis (SLE).

In another aspect of the invention, methods are provided for inhibiting MASP-2-dependent complement activation in a patient suffering from renal conditions including but not limited to mesangioproliferative agglomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis (mesangio-capillary glomerulonephritis) (post-streptococcal glomerulonephritis), cryoglobulinemic glomerulonephritis, lupus nephritis, purpura Henoch-Schonlein nephritis or IgA nephropathy.

In another aspect of the invention, methods are provided for inhibiting MASP-2-dependent complement activation in a patient suffering from a dermal condition, including but not limited to psoriasis, autoimmune bullous dermatoses, eosinophilic spongiosis, bullous pemphigoid, acquired bullous epidermolysis, and herpes. pregnancy and other dermal disorders or a thermal or chemical burn injury involving capillary effusion.

In another aspect of the invention, methods are provided for inhibiting MASP-2-dependent complement activation in a patient who has received an organ or other tissue transplantation, including but not limited to allograft or xenograft of organs (e.g. kidney, heart , liver, pancreas, lung, cornea, etc.) or grafts (eg, valves, tendons, bone marrow, etc.).

In another aspect of the invention, methods are provided for inhibiting MASP-2 dependent complement activation in a patient suffering from a central nervous system disorder or injury or a peripheral nervous system disorder or injury, including but not limited to multiple sclerosis (MS). ), myasthenia gravis (MG), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Guillain Barre syndrome, reperfusion following stroke, degenerative discs, brain trauma, Parkinson's disease (PD), Alzheimer's disease (AD), Miller-Fisher syndrome, brain trauma and / or hemorrhage, demyelination and meningitis.

In another aspect of the invention, methods are provided for inhibiting MASP-2 dependent complement activation in a patient suffering from a blood disorder including but not limited to septicemia or a condition resulting from septicemia including without limitation severe septicemia, septic shock, acute respiratory distress resulting from septicemia and systemic inflammatory response syndrome. Related methods are provided for the treatment of other blood disorders, including hemorrhagic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura (TTP), uremic hemolytic syndrome (HUS), or other destructive marrow / blood conditions.

In another aspect of the invention, methods are provided for inhibiting MASP-2-dependent complement activation in a patient suffering from a urogenital condition including but not limited to painful bladder disease, sensitive bladder disease, nonbacterial-chronic cystitis and interstitial cystitis, infertility. male and female, placental dysfunction and abortion and preeclampsia.

In another aspect of the invention, methods are provided for inhibiting MASP-2-dependent complement activation in a patient suffering from non-obese diabetes (Type 1 diabetes or insulin-dependent diabetes mellitus) or from complications of angiopathy, ouretinopathy, or diabetes mellitus neuropathy. Type 1 or Type 2 (onset in adulthood).

In another aspect of the invention, methods are provided for inhibiting MASP-2-dependent complement activation in a patient who is treated with chemotherapeutic products and / or radiation therapy, including without limitation for the treatment of cancerous conditions, by administering a drug inhibitor. MASP-2 to such a chemotherapeutic patient or radiation periterapy, that is, before and / or during and / or after administration of chemotherapeutic product (s) and / or radiation therapy. The administration of PERI chemotherapeutic product or radiation therapy to MASP-2 inhibitors may be useful for reducing the side effects of chemotherapeutic product or radiation therapy. In a still further aspect of the invention, methods are provided for treating malignancies by administering a deMASP-2 inhibiting agent in a pharmaceutically acceptable carrier to a patient suffering a malignancy.

In another aspect of the invention methods are provided for inhibiting MASP-2-dependent complement activation in a patient suffering from an endocrine disorder by administering a therapeutically effective amount of a MASP-2 inhibiting agent to a pharmaceutically acceptable carrier to such a patient. Conditions subjected to treatment according to the present invention include, by way of non-limiting example, Hashimoto's thyroiditis, stress, anxiety, and other potential hormonal disorders involving the regulated release of prolactin, depletion factor, or insulin-equivalent growth factor pituitary and adrenocorticotropin .

In another aspect of the invention methods are provided for inhibiting MASP-2 dependent complement activation in a patient suffering from age-related macular degeneration or other complement-mediated phthalmologic condition by administering a therapeutically effective amount of a MASP-2 inhibitory agent. in a pharmaceutical vehicle to a patient suffering from such a condition.

BRIEF DESCRIPTION OF DRAWINGS

The foregoing aspects and many of the attached advantages of this invention will become more readily appreciated as they become better understood by reference to the following detailed description, when considered in conjunction with the accompanying drawings, in which:

FIGURE 1 is a flowchart illustrating the new finding that the alternative complement pathway requires MASP-2 dependent activation of the lectin pathway for complement activation;

FIGURE 2 is a diagram illustrating the human MASP-2 genomic structure;

FIGURE 3A is a schematic diagram illustrating the domain structure of the human MASP-2 protein;

FIGURE 3B is a schematic diagram illustrating the domain structure of the human MAp 19 protein;

FIGURE 4 is a diagram illustrating the murine MASP-2 deletion strategy FIGURE 5 is a diagram illustrating the deminigene construct of human MASP-2;

FIGURE 6A presents results demonstrating that MASP-2 deficiency leads to loss of C4 activation mediated by lectin pathin as measured by lack of C4b deposition in mannan;

FIGURE 6B presents results demonstrating that MASP-2 deficiency leads to the loss of pathin-mediated C4 activation by lectin as measured by lack of C4b deposition in zymosan;

FIGURE 6C presents results demonstrating the relative C4 activation levels of serum samples obtained from MASP-2 +/-; MASP-2 - / - and wild-type strains as measured by C4b deposition in mannan and zymosan;

FIGURE 7A presents results demonstrating that MASP-2 deficiency leads to loss of C3 activation mediated by both lectin pathway and alternative pathway as measured by lack of C3b deposition in mannan;

FIGURE 7B presents results demonstrating that MASP-2 deficiency leads to loss of C3 activation mediated by both lectin pathway and alternative pathway as measured by lack of C3b deposition in zymosan;

FIGURE 8 presents results demonstrating that the addition of recombinant murine MASP-2 to MASP-2 - / - serum samples restores lectin pathway-mediated C4 activation in a manner dependent on protein concentration as measured by C4b deposition in mannan. ;

FIGURE 9 presents results demonstrating that the classical pathway is functional in the MASP-2 - / - strain;

FIGURE 10 presents results demonstrating that MASP-2-dependent complement system activation is activated in the ischemia / reperfusion phase following repair of the aortic-abdominal aneurysm;

FIGURE 11A shows results demonstrating that anti-MASP-2 # 11 Fab2 antibody inhibits C3 convertase formation, as described in Example 24;

FIGURE IIB shows results demonstrating that anti-MASP-2 # 11 Fab2 antibody binds to native rat MASP-2, as described in Example 24;

FIGURE IIC shows results demonstrating that anti-MASP-2 # 41 Fab2 antibody inhibits C4 cleavage, as described in Example 24;

FIGURE 12 presents results demonstrating that all tested anti-MASP-2 Fab2 antibodies that inhibited C3convertase formation were also found to inhibit C4 cleavage, as described in Example 24;

FIGURE 13 is a diagram illustrating rat MASP-2-derived recombinant polypeptides that were used for epitope mapping of anti-MASP-2 blocking Fab2 antibodies as described in Example 25;

FIGURE 14 shows results demonstrating anti-MASP-2 # 40 and # 60 Fab2 binding to rat MASP-2 polypeptides, as described in Example 25;

FIGURE 15 presents results demonstrating blood urea nitrogen uptake for wild type (+ / +) and MASP-2 (- / -) mice at 24 and 48 hours after reperfusion in a model of renal ischemia / reperfusion injury, as described in Example 26;

FIGURE 16A presents results demonstrating infarct size for wild type (+ / +) mice and reduced infarct size in MASP-2 (- / -) mice after injury in a coronary artery occlusion and reperfusion model as described. in Example 27;

FIGURE 16B presents results showing the distribution of individual animals tested in the coronary artery occlusion and reperfusion model as described in Example 27;

FIGURE 17A presents results showing reference VEGF protein levels in the wild-type (+ / +) mouse-type (+ / +) isolated RPE-choroid complex as described in

Example 28;

FIGURE 17B presents results showing VEGF protein levels in RPE-choroid complex on day 3 in wild type (+ / +) and MASP-2 (- / -) mice following laser-induced injury in a macular depletion model, such as described in Example 28;

FIGURE 18 presents results showing mean choroidal neovascularization (CNV) volume on day seven following laser-induced lesion in wild type (+ / +) and MASP-2 (- / -) mice, as described in Example 28;

FIGURE 19 presents results showing the mean clinical wild-type (+ / +) and MASP-2 (- / -) mouse count with time following mAb Col2-induced rheumatoid arthritis, as described in Example 29;

FIGURE 20A is a diagram showing the targeted disruption of the sMAP gene (Mapl9) as described in Example 30;

FIGURE 20B shows the Southern blot analysis of isolated progeny genomic DNA derived from mating chimeric male (- / -) mice with female C57BL / 6 mice, as described in Example 30;

FIGURE 20C shows the PCR genotyping analysis of wild type (+ / +) and sMAP (- / -) mice, as described in Example 30;

FIGURE 21A shows Northern blot analysis of sMAP and MASP-2 mRNAs in sMAP (- / -) mice, as described in Example 30;

FIGURE 2IB presents the quantitative cDNA RT-PCR analysis encoding MASP-2α-chain, MASP-2L-chain and sMAP, in wild type (+ / +) mice and sMAP (- / -), as described in the Example 30;

FIGURE 22A shows a sMAP (- / -) immunoblot, i.e., MAp 19 (- / -), which demonstrates MASP-2 and sMAP deficiency in mouse serode as described in Example 30;

FIGURE 22B shows results demonstrating that MASP-2 and sMAP were detected in the MBL-MASP-sMAP complex, as described in Example 30;

FIGURE 23A shows results showing deposition of C4 in manna-coated reservoirs in wild type (+ / +) and sMAP (- / -) mouse as described in Example 30;

FIGURE 23B presents results showing deposition of C3 in manna coated reservoirs in wild type (+ / +) and sMAP (- / -) mouse as described in Example 30;

FIGURE 24A presents results showing the reconstitution of MBL-MASP-sMAP complex in sMAP (- / -) serum, as described in Example 30;

FIGURES 24B to D show results showing competitive allocation of rsMAP and MASP-21 to MBL, as described in Example 30;

Figures 25A and B show results showing restoration of C4 deposition activity by addition of rMASP-2, but not rsMAP as described in Example 30. Figures 26A and B show results showing reduction of C4 deposition activity by addition of rsMAP as described in Example 30; and

FIGURES 27A and C show results showing that MASP-2 is responsible for activation by C4 shift of C3, as described in Example 31.

DESCRIPTION OF SEQUENCE LIST

SEQ ID NO: 1 cDNA of human MAp 19

SEQ ID NO: 2 human MAp 19 protein (with leader)

SEQ ID NO: 3 human (mature) MAp 19 protein

SEQ ID NO: 4 Human MASP-2 cDNA

SEQ ID NO: 5 Human MASP-2 Protein (with leader)

SEQ ID NO: 6 human (mature) MASP-2 protein

SEQ ID NO: 7 gDNA OF HUMAN MASP-2 (exons 1 to 6) ANTIGENS: (REFERENCE TO MATURE MASP-2 PROTEIN)

SEQ ID NO: 8 CUBI sequence (amino acids 1 to 121)

SEQ ID NO: 9 CUBEGF sequence (amino acids 1 to 166)

SEQ ID NO: 10 CUBEGFCUBII (amino acids 1 to 293)

SEQ ID NO: 11 EGF region (amino acids 122 to 166)

SEQ ID NO: 12 serine protease domain (amino acids 429a 671)

SEQ ID NO: 13 inactive serine protease domain (amino acids 610 to 625 with mutation from Ser618 to Ala)

SEQ ID NO: 14 TPLGPKWPEPVFGRL (CUB 1 peptide)

SEQ ID NO: 15 TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQ (CUBI Peptide)

SEQ ID NO: 16 TFRSDYSN (MBL Binding Region Core)

SEQ ID NO: 17 FYSLGSSLDITFRSDYSNEKPFTGF (MBL Binding Region)

SEQ ED NO: 18 EDECQVAPG (EGF PEPTIDE) SEQ ID NO: 19 ANMLCAGLESGGKDSCRGDSGGV (Serine Protease Binding Core) Detailed Description PEPTIDE INHIBITORS:

SEQ ID NO: 20 MBL Life Size cDNA

SEQ ID NO: 21 MBL Life Size Protein

SEQ ID NO: 22 OGK-X-GP (Consensus Link)

SEQ ID NO: 23 OGKLG

SEQ ID NO: 24 GLR GLQ GPO GKL GPO G

SEQ ID NO: 25 GPO GPO GLR GLQ GPO GKL GPO GPO

GPO

SEQ ID NO: 26 GKDGRDGTKGEKGEPGQGLRGLQG

POGKLGPOG

SEQ ID NO: 27 GAOGSOGEKGAOGP QGPOGPOGKMGPKGEOGD O (human h-ficoline)

SEQ ID NO: 28 GCOGLOGAOGDKGEAGTNGKRGERGPOGPOGK AGPOGPN GAOGEO (human p35 ficoline)

SEQ ID NO: 29 LQRALEILPNRVTIKANRPFLVFI (C4 slope site)

EXPRESSION INHIBITORS:

SEQ ID NO: 30 cUBI-EGF domain cDNA (nucleotides 22 to 680 of SEQ ID NO: 4)

SEQ ID NO: 31

5 'CGGGCACACCATGAGGCTGCTGACCCTCCTGGGC

3 '

Nucleotides 12 to 45 of SEQ ID NO: 4 including site of translation start of MASP-2 (sense) SEQ ID NO: 325'GAC ATTACCTTCCGCTCCGACTCC AACGAGAAG3'Nucleotides 361 to 396 of SEQ ED NO: 4 encoding a region comprising the site MBL connection cable (direction)

SEQ ID NO: 33

5'AGCAGCCCTGAATACCCACGGCCGTATCCCAAA3 '

Nucleotides 610 to 642 of SEQ LD NO: 4 encoding a region comprising the CLONING CUBITINICIATORS domain:

SEQ ID NO: 34 CGGGATCCATGAGGCTGCTGACCCTC (5 'PCR FOR CUB)

SEQ ID NO: 35 GGAATTCCTAGGCTGCATA (3 'PCRPARA CUB)

SEQ ID NO: 36 GGAATTCCTACAGGGCGCT (3 'PCRPARA CUBIEGF)

SEQ ID NO: 37 GGAATTCCTAGTAGTGGAT (3 'PCRPARA CUBIEGFCUBII)

SEQ ID NOS: 38 to 47 are humanized paraantibody cloning primers SEQ ID NO: 48 is 9 amino acid peptide binding

EXPRESSION VECTOR:

SEQ ID NO: 49 is the MASP-2 minigene insert

SEQ ID NO: 50 is murine MASP-2 cDNA

SEQ ID NO: 51 is murine MASP-2 protein (w / leader)

SEQ ID NO: 52 is mature murine MASP-2 protein

SEQ ID NO: 53 The Mouse MASP-2 cDNA

SEQ ID NO: 54 is rat MASP-2 protein (w / leader)

SEQ ID NO: 55 is mature rat MASP-2 protein

SEQ ID NO: 56 to 59 are the oligonucleotides for human MASP-2 site-directed mutagenesis used to generate human MASP-2A

SEQ ID NO: 60 to 63 are the murine MASP-2 site directed oligonucleotides used to generate murine MASP-2A

SEQ ID NO: 64 to 65 are the mouse MASP-2 site directed oligonucleotides used to generate mouse MASP-2A

DETAILED DESCRIPTION

The present invention is based on the surprising findings by the present inventors that MASP-2 is necessary to initiate activation of the alternative complement pathway. Through the use of a dead mouse model of MASP-2 - / -, the present inventors have shown that it is possible to inhibit the complement complement pathway activation via the lectin-mediated MASP-2 pathway while leaving the classical pathway intact, thus establishing the activation of the MASP-2 - / - mouse. Lectin-dependent as a requirement for alternate complement activation in the absence of the classical pathway. The present invention also describes the use of MASP-2 as a therapeutic target to inhibit cell damage associated with lectin-mediated alternative complement pathway activation while leaving the Clq-dependent truck component of the immune system intact.

I. DEFINITIONS

Unless specifically defined herein, all terms used herein have the same meaning as would be understood by those of ordinary skill in the art of the present invention. The following definitions are provided in order to provide clarity with respect to the terms such as those used in the specification and claims to describe the present invention.

As used herein, the term "MASP-2 dependent complement activation" refers to the alternative pathway complement activation that occurs through delectin dependent MASP-2 activation.

As used herein, the term "alternative pathway" refers to complement activation that is triggered by, for example, zymosan yeast and fungal cell walls, lipopolysaccharides (LPS), Gram negative external membranes and rabbit erythrocytes, as well as very pure depolysaccharides. , rabbit erythrocytes, viruses, bacteria, animal tumor cells, parasites and damaged cells and which has traditionally been considered to arise from spontaneous proteolytic generation of C3b from complement factor C3.

As used herein, the term "lectin pathway" refers to complement activation that occurs through specific binding of serum and non-serum carbohydrate-binding proteins including lectin chelated mannan (MBL) and phytolins.

As used herein, the term "classical pathway" refers to complement activation that is triggered by the antibody bound to a foreign particle and requires the binding of the Clq recognition molecule.

As used herein, the term "MASP-2 inhibitory agent" refers to any agent that binds to or directly interacts with MASP-2 and effectively inhibits MASP-2-dependent complement activation, including anti-MASP-2 antibodies. 2 and MASP-2 binding fragments of these, natural and synthetic peptides, small molecules, soluble MASP-2 receptors, expression inhibitors and isolated natural inhibitors and also peptides competing with MASP-2 for binding to another recognition molecule (eg MBL, H-ficoline, M-ficoline or L-ficoline) in the lectin pathway, but do not encompass antibodies that bind to such other recognition molecules. MASP-2 inhibitory agents useful in the method of the invention may reduce MASP-2 dependent complement activation by more than 20%, such as more than 50%, such as more than 90%. In one embodiment, the MASP-2 inhibitory agent reduces MASP-2-dependent complement activation by more than 90% (that is, resulting in only 10% or less deMASP-2 complement activation).

As used herein, the term "antibody" encompasses antibodies and antibody fragments thereof, derived from any antibody-producing mammal (e.g., mouse, rat, rabbit and primate including human), which specifically bind to MASP-2 polypeptides or portions thereof. . Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; multispecific antibodies (e.g., bispecific antibodies); humanized antibodies; murine antibodies: chimeric monoclonal antibodies, mouse, primate mouse, human primate; and anti-idiotype antibodies and may be any intact molecule or fragment thereof.

As used herein, the term "antibody fragment" refers to a portion derived from or related to a naturally-sized anti-MASP-2 antibody, generally including antigen binding or variable region thereof. Illustrative examples of antibody fragments include Fab, Fab ', F (ab) 2, F (ab') 2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

As used herein, a "single chain Fv" or "scFv" antibody fragment comprises the V H and V L domains of an antibody, where these domains are present on a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains, which allows the scFv to form the desired structure for antigen binding.

As used herein, a "chimeric antibody" is a recombinant protein that contains the variable domains and complementarity determining regions derived from a non-human species antibody (e.g., rodent), while the rest of the antibody molecule is derived from a human antibody.

As used herein, a "humanized antibody" is a chimeric antibody that comprises a minimal sequence that conforms to specific complementarity determining regions derived from non-human immunoglobulin that is transplanted into a human antibody matrix. Humanized antibodies are typically recombinant proteins wherein only complementarity determining antibody regions are of non-human origin.

As used herein, the term "mannan binding lectin" ("MBL") is equivalent to the mannan binding protein ("MBP").

As used herein, the "membrane attack complex" ("MAC") refers to a complex of the five terminal complement components (C5-C9) that inserts into and ruptures membranes. Also alluded to as C5b-9.

As used herein, "one patient" includes all mammals, including without limitation humans, nonhuman primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.

As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), Glutamine (Gln; Q), Glycine (Gly; G), Histidine (His; H), Isoleucine (Ile; I), Leucine (Leu; L), Lysine (Lys; K), Methionine ( Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and evaline (Val; V).

In the broadest sense, naturally occurring amino acids can be divided into groups based on the side-chain chemical characteristics of the respective amino acids. By "hydrophobic" amino acid is meant Ile, Leu, Met, Phe3 Trp5 Tyr, Val, Ala, Cys or Pro. By "hydrophilic" amino acid is meant Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His. This amino acid grouping may further be subclassified as follows. By "uncharged hydrophilic" amino acid is meant Ser, Thr, Asn or Gln. By amino acid "acid" is meantGlu or Asp. By "basic" amino acid is meant Lys, Arg or His.

As used herein the term "conservative amino acid substitution" is illustrated by an amino acid substitution within each of the following groups: (1) glycine, alanine, valine, leucine and isoleucine, (2) phenylalanine, tyrosine and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine and (6) lysine, arginine and histidine.

The term "oligonucleotide" as used herein refers to a ribonucleic acid (RNA) or polymer or deoxyribonucleic acid (DNA) polymer or mimetic thereof. This term also encompasses those oligonucleobases composed of nucleotide, sugars, and covalent (main chain) internucleoside linkages as well as oligonucleotides having non-naturally occurring modifications.

II. The Alternative Way: A New Understanding

The alternative complement pathway was first described by Louis Pillemer and his colleagues in the 1950s based on studies in which zimosan made from yeast cell walls was used to activate complement (Pillemer, L. et al., J. Exp. Med. 103: 1-13, 1956; Lepow, 1H, J. Immunol. 125: 471-478 (1980). Zimosan has since been considered as the canonical example of a specific alternative pathway activator in human and rodent serum (Lachmann, PJ, et al., Springer Semin. Immunopathol. 7: 143-162, 1984; Van Dijk, H., et J. Immunol Methods85: 233-243 (1985; Pangburn, K., Metods in Enzymol. 162: 639-653,1988). A convenient and widely used assay for alternate pathway activation is to incubate serum with coated zymosan in plastic reservoirs and determine the amount of C3b deposition on the solid phase following incubation. As expected, there is substantial deposition of C3 over zimosan-coated reservoirs following normal mouse serum incubation (Figure 7B). However, incubating serum from homozygous MASP-2 deficient mice with zimosan-coated reservoirs results in a substantial reduction in C3b deposition compared with that of normal serum. In addition, the use of MASP 2 gene deficient heterozygous mouse serum in this assay results in C3b deposition levels that are intermediate between those obtained with MASP-2 deficient mouse homozygous mouse serum serum. Parallel results are also obtained using mannan-coated reservoirs, another polysaccharide known to activate the alternative pathway (Figure 7A). Since normal and MASP-2 deficient mice share the same genetic background except for the MASP 2 gene, these unexpected results demonstrate that MASP-2 plays an essential role in activating the alternative pathway.

These results provide strong evidence that the alternative pathway is not an independent, autonomous complement activation pathway as described essentially in all current medical textbooks and recent review articles on complement. The current and widely held scientific view is that the alternative pathway is activated on the surface of certain particulate targets (microbes, zymosan, red blood cells) through amplification of spontaneous C3 iiUck-Over activation. However, the absence of activation of the significant alternative pathway in serum. of dead mice in MASP-2 by two well-known "activators" of the alternate pathway makes it unlikely that the "tick-over" theory will describe an important physiological mechanism for decomposition activation.

Since MASP-2 protease is known to have a specific and well-defined role as the enzyme responsible for lectin complement initiation, these results imply the activation of lectin pathway by zimosan and mannan as a critical first step for subsequent activation of lectin. Alternative way. C4b is a reactivation product generated by the lectin pathway but not the alternative pathway. Compatible with this concept, incubation of normal mouse serum with zimosan or mannan coated reservoirs results in deposition of C4b on the reservoirs and this deposition of C4b is substantially reduced when Coated reservoirs are incubated with MASP-2 deficient mouse serum (Figures 6A, 6B and 6C).

The alternative pathway, in addition to its widely accepted role as an independent pathway for complement activation, may also provide an amplification loop for initially triggered complement activation via the classical and lectin pathways (Liszewski, Μ. K. and JP Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by WE Paul, Raven Press, Ltd., New York; Schweinie, JE, et al., J. Clin. Invest. 84: 1821-1829, 1989). In this alternative pathway-mediated amplification mechanism, C3convertase (C4b2b) is generated by activation of the C3 complementoclassical or lectin cleavage cascades at C3a and C3b and thus provides C3b which can participate in the formation of C3bBb, the C3 convertase of the alternate pathway. The likely explanation for the absence of alternate pathway activation in dead serum in MASP-2 is that the lectin pathway is required for initial complement activation by zimosan, manana and other putative "activators" of the alternative pathway, while the alternative pathway plays a crucial role. for amplification of complement activation. In other words, the alternative path is a feed-amplifying loop dependent on the delectin and classic complement pathways for activation, rather than a linear independent cascade.

Instead of the complement cascade that is activated via three distinct pathways (classical, alternative and lectin pathways) as previously predicted, our results indicate that it is more accurate to view complement as being composed of two main systems, which correspond, at first glance, to the innate (lectin) and acquired (classical) alas of the complement immune defense system. Lectins (MBP, M-Ficoline, H-Ficoline and L-Ficoline) are the specific recognition molecules that trigger the innate complement system and the system includes the lectin pathway and the associated alternative pathway amplification loop. Clq is the specific recognition molecule that triggers the acquired complement system and the system includes the classic truck and associated alternative path amplification loop. We refer to these two major complement activation systems as the lectin dependent complement system and the complement complement dependent Clq system, respectively.

In addition to its essential roles in immune defense, the breakdown system contributes to tissue damage in many clinical conditions. Thus, there is a pressing need to develop therapeutically effective breakdown inhibitors to prevent these adverse effects.

With the recognition that complement is comprised of two major complement activation systems, it is realized that it would be highly desirable to specifically inhibit only the complement activation system that causes a particular pathology without completely paralyzing complement immune defense capabilities. For example, in disease states in which complement activation is predominantly mediated by the lectin-dependent complement system, it would be advantageous to specifically inhibit only this system. This would leave the Clq-dependent complement activation system intact to deal with immune complex processing and aid in host defense against infection.

The preferred protein component for targeting the development of therapeutic agents to specifically inhibit lectin-dependent complement system is MASP-2. Of all the protein components of the lectin-dependent complement system (MBL, H-ficoline, M-ficoline, L-ficoline, MASP-2, C2-C9, Factor B, Properdin Factor), only MASP-2 is unique. for the lectin-dependent supplement system as required for the system to function. Lectins (MBL, H-Ficoline, M-Ficoline and L-Ficoline) are also unique components in the lectin-dependent complement system. However, the loss of any of the lectin components would not necessarily inhibit system activation due to lectin redundancy. It would be necessary to inhibit all four lectins to ensure inhibition of activation of the lectin dependent complement system. In addition, since MBL and phytolins are also known to have complement-independent opsonic activity, inhibition of lectin function would result in the loss of the host's defense defense against infection. In contrast, this complement-independent lectin opsonic activity would remain intact if MASP-2 were the inhibitory target. An additional benefit of MASP-2 as the therapeutic target for inhibiting lectin-dependent complement system activation is that the plasma concentration of MASP-2 is among the lowest of any protein-depleting complement (≤ 500 ng / ml); therefore correspondingly low concentrations of high affinity inhibitors of MASP-2 may be required to achieve complete inhibition (Moller-Kristensen, M., et al., J. ImmunolMethods 282: 159-167, 2003).

III. MASP-2 ROLE IN VARIOUS DISEASES AND THERAPEUTIC METHODS USING MASP-2 INHIBITORY AGENTS

INJURY BY ISOUEMIC REPERFUSION

Ischemic reperfusion injury (I / R) occurs when blood flow is restored after an extended period of ischemia. It is a common source of morbidity and mortality in a broad spectrum of diseases. Surgical patients are vulnerable after repair of aortic deaneurysm, cardiopulmonary bypass, vascular reanastomosis and connection with, for example, organ transplants (eg, heart, lung, liver, kidney) and finger / extremity reimplantation, stroke, myocardial infarction and hemodynamic resuscitation following shock and / or surgical procedures. Patients with atherosclerotic diseases are prone to myocardial infarctions, strokes, and embolus-induced intestinal and lower extremity ischemia. Trauma patients often suffer from temporary limb ischemia. Also, any cause of massive blood loss leads to a full body I / R reaction.

The pathophysiology of the I / R lesion is complex, with at least two major factors contributing to the process: complement activation and neutrophil stimulation with the accompanying oxygen-mediated lesion. In I / R injury, complement activation was first described during myocardial infarction over 30 years ago and has led numerous investigations into the contribution of the complement system to I / R tissue injury (Hill, JH, et al., J. Exp. Med. 133: 885-900,1971). Cumulative evidence now points to complement as a central mediator in I / R injury. Complement inhibition has been successful in lesion limitation in several animal models of I / R. In the initial studies, C3 depletion was obtained following snake venom factor infusion, reported to be beneficial during kidney and heart I / R (Maroko, PR, et al., 1978, J. Clin Invest. 61: 661- 670, 1978; Stein, SH, et al., Miner Electrolyte Metab. 11: 256-61 (1985). However, the soluble complement receptor form 1 (sCR1) was the first specific inhibitor of complement used for the prevention of myocardial I / R injury (Weisman, H.F., et al., Science 249: 146-51, 1990). Treatment for myocardial I / R attenuates the infarction associated with decreased deposition of C5b-9 complexes along the coronary endothelium and decreased leukocyte infiltration after reperfusion.

In experimental myocardial I / R, the Cl esterase inhibitor (ClINH) administered before reperfusion prevents Clq deposition and significantly reduced the area of cardiac muscle necrosis (Buerke, M., et al., 1995, Circulation 91: 393- 402, 1995). Genetically C3-deficient animals have less local tissue necrosis after skeletal or intestinal, ischemia (Weiser, M.R., et al., J. Exp. Med. 183: 2343-48, 1996).

Membrane attack complex is the last complement-directed lesion vehicle, and studies in C5-deficient animals have shown decreased local and remote lesion in I / R injury models (Austen, WG Jr., et al., Surgery 126: 343- 48, 1999). A breakdown activation inhibitor, soluble Crry (breakdown receptor-related Y gene) has been shown to be effective against injury when given both before and after the onset of murine intestinal reperfusion (Rehrig, S., et al., J. Immunol. 167 : 5921-27, 2001). In a model of skeletal muscle ischemia, the use of soluble complement receptor 1 (sCRl) also reduced muscle injury when given after reperfusion onset (Kyriakides, C., et al., Am. J. Physiol. Cell Physiol. 281 : C244-30, 2001). In a porcine myocardial I / R model, anti-monoclonal ("MoAb") treated animals for anaphylatoxin C5a prior to reperfusion showed attenuated infarction (Amsterdam, A.A., et al., Am. J. Physiol. HeartCirc. Physiol. 268: H448-57, 1995). C5 MoAbd-treated rats demonstrated attenuated infarct size, neutrophil infiltration, and myocardial apoptosis (Vakeva, A., et al., Circulation 97: 2259-67, 1998). These experimental results highlight the importance of decomposition activation in the pathogenesis of lesion I / R.

It is uncertain that the complement pathway (classical, lectin or alternative) is predominantly involved in the decomposition activation in the I / R lesion. Weiser et al. demonstrated an important role in lectin and / or classical pathways during skeletal I / R showing that dead C3 or C4 mice were protected against I / R injury based on a significant reduction in vascular permeability (Weiser, MR, et al., J Exp. Med. 183: 2343-48, 1996). In contrast, renal I / R experiments with C4 inoperative mice showed no significant tissue protection, while C3, C5, and C6 dead mice were protected from injury, suggesting that activation of complement during renal I / R injury occurs via the alternate pathway. (Zhou, W., et al., J. Clin. Invest. 105: 1363-71, 2000). Using factor D deficient mice, Stahl et al. have recently shown evidence for an important role of the alternative pathway in intestinal I / R in mice (Stahl, G., et al., Am. J. Pathol. 162: 449-55, 2003). In contrast, Williams et al. have suggested a predominant role of the classic truck for the initiation of I / R lesion in the mouse intestine showing reduced organ damage to C3 and injury protection in C4 and IgM deficient mice (Ragl - / -) (Williams, JP, et al., J Appl. Physiol., 86: 938-42, 1999).

Treatment of rats in a myocardial I / R model with monoclonal mannan-binding rat lectin (MBL) antibodies resulted in reduced post-ischemic reperfusion injury (Jordan, J.E., et al., Circulation 104: 1413-18, 2001). MBL antibodies also reduced complement uptake in endothelial cells in vitro after oxidative stress indicating a role for the lectin pathway in I / Myocardial injury (Collard, CD, et al., Am. J. Pathol. 156: 1549-56, There is also evidence that I / R injury in some organs may be mediated by a specific IgM category called natural antibodies and classic pathway activation (Fleming, SD, et al., J. Immunol. 169: 2126- 33, 2002; Reid, RR, et al., J. Immunol. 169: 5433-40, 2002).

Several complement activation inhibitors have been developed as potential therapeutic agents to prevent morbidity and mortality resulting from myocardial I / R complications. Two of these inhibitors, humanized sCR1 (TP10) and anti-05 scFv (Pexelizumab), completed Phase II clinical trials. Pexelizumab additionally completed a Phase III clinical trial. Although TP10 was well tolerated and beneficial to patients at the onset of Phase I / II testing, results from a Phase II test completed in February 2002 failed to reach its primary pontofinal. However, subgroup analysis of data from male patients in a high-risk population undergoing open-heart procedures showed significantly decreased mortality and infarct size. Administration of an antihumanized scFv decreased overall patient mortality associated with acute myocardial infarction in the Phase II COMA and COMPLY tests, but failed to reach the primary end point (Mahaffey, KW, et al., Circulation 108: 1176- 83, 2003). Results from a recent Phase III anti-05 scFv clinical trial (PRIMO-CABG) to improve the surgically induced consequences following coronary artery bypass were recently released. Although the primary end point for this study was not reached, the studies demonstrated an overall reduction in postoperative patient morbidity and morbidity.

Dr. Walsh and colleagues demonstrated that mice lacking MBL and therefore lacking MBL-dependent dalectin pathway activation but with fully active classical complement pathways are protected from cardiac reperfusion injury with preservation resulting from cardiac function (Walsh et al. J. Immunol 175: 541-46, 2005). Significantly, mice lacking Clq, the classic complement pathway recognition component that has intact MBL complement pathway, are not protected from injury. These results indicate that the lectin pathway plays a major role in the pathogenesis of ischemic myocardial reperfusion injury. .

Complement activation is known to play an important role in tissue damage associated with gastrointestinal ischemia-reperfision (I / R). Using a murine model of GI / R, a student by Hart and colleagues reports that genetically MBL-deficient mice are protected from intestinal injury after gastrointestinal I / R (Hartet al., J. Immunol. 174: 6373-80, 2005). . The addition of recombinant MBL to MBL deficient mice significantly increased the lesion compared to untreated MBL deficient mice after I / R gastrointestinal. In contrast, mice that genetically lack Clq, the classic pathway recognition component, are not protected from tissue damage after gastrointestinal I / R.

Renal I / R is a major cause of acute renal failure. The complement system appears to be essentially involved in renal Iales by I / R. In a recent study by Vries and colleagues report that the lectin pathway is activated in the course of experimental renal injury as well as clinical I / R (de Vries et al., Am. J. Path. 165: 1677-88, 2004). In addition, the lectin pathway precedes and co-localizes with the deposition of C3, C6 and C9 complement in the course of renal I / R. These results indicate that the dalectin pathway of complement activation is involved in renal I / R injury.

One aspect of the invention is thus directed to the treatment of ischemic reperfusion deletion by treating a patient experiencing ischemic perfusion with a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier. The inhibitor of DEMASP-2 may be administered to the patient by intraarterial, intravenous, intracranial, intramuscular, subcutaneous or other parenteral administration and potentially orally to non-peptidergic inhibitors and more suitably by intraarterial or intravenous administration. Administration of the MASP-2 inhibitor compositions of the present invention suitably begins immediately after or as soon as possible after an ischemic reperfusion event. In cases where reperfusion occurs in a controlled environment (for example, following repair of aortic aneurysm, organ transplantation, or reconnected or traumatized limb or fingers), the MASP-2 inhibitory agent may be administered before and / or during and / or after reperfusion. Administration may be repeated periodically as determined by a physician for optimal therapeutic effect.

Atherosclerosis

There is considerable evidence that complement activation is involved in atherogenesis in humans. Several studies have convincingly shown that while no significant complement activation occurs in normal arteries, complement is extensively activated in atherosclerotic lesions and is especially strong in ruptured and vulnerable plates. Components of the complementoterminal pathway are often found in human atheromas (Niculescu, F., et al., Mol. Immunol. 36: 949-55, 10-12, 1999; Rus, HG, et al., Immunol. Lett. 20: 305-310, 1989; Torzewski, M., et al., Arterioscler (Thromb. Vasc. Biol. 18: 369-378, 1998). Deposition of C3 and C4 in arterial lesions has also been demonstrated (Hansson, G.K., et al., Acta Pathol.Microbiol. Immunol. Scand. (A) 92: 429-35, 1984). The degree of deposition of C5b-9 has been found to correlate with the severity of the lesion (Vlaicu, R., etal., Atherosclerosis 57: 163-77, 1985). Completion deposition of iC3b, but not of C5b-9, was especially strong in ruptured and vulnerable plaques, suggesting that complement activation may be a factor in acute syndromesoronary syndromes (Taskinen S., et al., Biochem. J. 367: 403-12, 2002). Experimental emateroma in rabbits, complement activation has been found to precede lesion development (Seifer, P. S., et al., Lab Invest. 60: 747-54, 1989).

In atherosclerotic lesions, complement is activated via the classical and alternative pathways, but there is little evidence so far of complement activation via the lectin pathway. Several components of the arterial wall may trigger the activation of complement. The classical complement pathway can be activated by enzymatically degraded LDL-linked C-reactive protein (CRP) (Bhakdi, S., et al., Arterioscler. Thromb. Vasc. Biol. 19: 2348-54, 1999). Compatible with this theory is the discovery that complementoterminal proteins co-localize with CRP within early human lesions (Torzewski, J., et al., Arterioscler. Thromb. Vasc. Biol. 18: 1386-92, 1998). Similarly, oxidized LDL-specific immunoglobulin M or IgG antibodies within lesions may activate the classical pathway (Witztum, JL, Lancet 344: 793-95, 1994). Lipids isolated from human atherosclerotic lesions have a high non-esterified cholesterol content and are capable of reactivating the alternative pathway (Seifert P. S., et al., J. Exp. Med. 172: 547-57,1990). Chlamydia pneumoniae, a Gram negative bacterium often associated with atherosclerotic lesions, can also activate the alternative complement pathway (Campbell L. A., et al., J. Infect. Dis. 172: 585-8,1995). Other potential complement activators present in atherosclerotic lesions include cholesterol crystals and cell debris, both of which may activate the alternative pathway (Seifert, P. S., et al., Mol. Immunol. 24: 1303-08, 1987).

By-products of complement activation are known to have many biological properties that could influence the development of atherosclerotic lesions. Local complement activation can induce cell lysis and generate at least some of the cell fragments found in the necrotic nucleus of advanced lesions (Niculescu, F. et al., Mol. Immunol. 36: 949-55, 10-12, 1999). Activation of sublimate complement may be a significant factor contributing to smooth muscle cell proliferation and monocyte infiltration into the arterial intima during atherogenesis (Torzewski J., et al., Arterioscler. Thromb. Vasc. Biol.18: 673-77 , 1996). Persistent complement activation can be harmful because it can trigger and sustain inflammation. In addition to the decomposing complement infiltration of blood plasma, arterial cells express messenger RNA to complement proteins, and expression of various complement components is over-regulated in atherosclerotic lesions (Yasojima, K., et al., Arterioscler. Thromb. Vasc. Biol 21: 1214-19, 2001).

A limited number of studies on the influence of complement protein deficiencies on atherogenesis have been reported. Results in experimental animal models were conflicting. In rats, the formation of toxic atherosclerotic equivalent doses induced by devitamin D was decreased in complement depleted animals (GeertingerP., Et al., Acta. Pathol. Microbiol. Scand. (A) 78: 284-88, 1970). In addition, in cholesterol-fed rabbits, inhibition of complement by genetic C6 deficiency (Geertinger, P., et al., Artery 1: 177-84, 1977; Schmiedt, W., et al., Arterioscl. Thromb. Vasc Biol. 18: 1790-1795, 1998) or by the K-76 COONa anti-complement agent (Saito, E., et al., J. Drug Dev. 3: 147-54, 1990) suppressed the development of atherosclerosis without affecting the levels of serum cholesterol. In contrast, a recent study reported that C5 deficiency does not reduce the development of atherosclerotic lesions in apolipoprotein E (ApoE) deficient mice (Patel, S., et al., Biochem. Biophys. Res. Commun. 286: 164-70, 2001). However, in another study the development of atherosclerotic lesions in LDLR-deficient mice (ldlr-) with or without C3 deficiency was evaluated (Buono, C., et al., Circulation 105: 3025-31, 2002). They found that atheroma maturation for atherosclerotic-equivalent lesions depends in part on the presence of an intact complement system.

One aspect of the invention is thus directed to the treatment or prevention of atherosclerosis by treating a patient suffering from or prone to atherosclerosis with a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier. The MAPS-2 inhibitory agent may be administered to the patient by intraarterial, intravenous, intrathecal, intracranial, intramuscular, subcutaneous or other parenteral administration and potentially orally to non-peptidergic inhibitors. Administration of the MASP-2 inhibitor composition may begin after the diagnosis of atherosclerosis in a patient or in a patient at high risk of developing such a condition. Administration may be repeated periodically as determined by a physician for the optimal therapeutic effect.

OTHER VASCULAR DISEASES AND CONDITIONS

The endothelium is largely exposed to the immune system and is particularly vulnerable to complement proteins that are present in plasma. Complement-mediated vascular injury has been shown to contribute to the pathophysiology of several diseases of the cardiovascular system, including atherosclerosis (Seifert, PS, et al., Atherosclerosis 73: 91-104,1988), ischemia-reperfusion injury (Weisman, HF , Science 249: 146-51,1990) and myocardial infarction (Tada, T., et al., Virchows Arch 430: 327-332,1997). Evidence suggests that complement activation may extend to other vascular conditions.

For example, there is evidence that complement activation contributes to the pathogenesis of many forms of vasculitis, including: Henoch-Schonlein purpura nephritis, vasculitis associated with systemic olupus erythematosus, vasculitis associated with rheumatoid arthritis (also called malignant rheumatoid arthritis) , immune complex vasculitis and Takayasu disease. Henoch-Schonlein purpura nephritis is a form of systemic vasculitis of small vessels with immune pathogenesis, where complement activation via the lectin pathway leading to C5b-9-induced endothelial molting is recognized as an important mechanism (Kawana, S., et al. Arch. Dermatol Res. 282: 183-7, 1990; Endo, M., et al., Am J. Kidney Dis. 35: 401-7, 2000). Systemic lupus erythematosus (SLE) is an example of systemic autoimmune diseases affecting multiple organs, including skin, kidneys, joints, serosal surfaces, and central nervous system and is often associated with severe vasculitis. Anti-endothelial IgG antibodies and IgG complexes capable of binding to endothelial cells are present in the sera of patients with active SLE, and immune and complement complement IgG deposits are found in the blood vessel walls of patients with SLE vasculitis (Cines, DB, et al. , J. Clin.Invest., 73: 611-25, 1984). Rheumatoid arthritis associated with vasculitis, also called malignant rheumatoid arthritis (Tomooka, K., Fukuoka Igaku Zasshi 80: 456-66, 1989), immune complex vasculitis, associated with hepatitis A, leukocytoclastic vasculitis, and known arteritis as Takayasu, form another pleomorphic group of human diseases in which complement-dependent cytotoxicity against endothelial and other cell types plays a documented role (Tripathy, NK, et al, J. Rheumatol. 28: 805-8, 2001).

Evidence also suggests that complement activation plays a role in dilated cardiomyopathy. Dilated cardiomyopathy is a syndrome characterized by cardiac enlargement and impaired systolic function of the heart. Recent data suggest that early myocardial inflammation may contribute to the development of disease. C5b-9, the terminal complement membrane attack complex, is known to correlate significantly with TNF-alpha myocardial expression and myocardial deposition. In myocardial biopsies of 28 patients with dilated cardiomyopathy, C5b-9 accumulation has been shown, suggesting that chronic immunoglobulin-mediated activation activation in the myocardium may partly contribute to the progression of dilated cardiomyopathy (Zwaka, TP, et al., Am. J Pathol 161 (2): 449-57, 2002).

One aspect of the invention is thus directed to the treatment of a vascular condition, including cardiovascular conditions, cerebrovascular conditions, peripheral vascular conditions (e.g., skeletal muscle), renovascular conditions, and mesenteric / enteric vascular conditions, by administering a composition comprising a therapeutically effective amount of an agent. MASP-2 inhibitor in a pharmaceutical carrier. Conditions for which the invention is believed to be adapted include, but are not limited to: vasculitis, including Henoch-Schonlein purpura nephritis, vasculitis associated with systemic lupuseritis, rheumatoid arthritis-associated vasculitis (also called malignant rheumatoid arthritis), vasculitis of immune complex Takayasu; dilated cardiomyopathy; diabetic angiopathy; Kawasaki disease (arteritis); and poison gas plunger (VGE). Also, since complement activation occurs as a result of luminal trauma and inflammatory foreign body response associated with cardiovascular intervention procedures, it is believed that the DEMASP-2 inhibitor compositions of the present invention may also be used for post-stenosis inhibition. Probe, rotational atherectomy and / or percutaneous transluminal coronary angioplasty (PTCA), alone or in combination with other restenosis inhibiting agents such as are disclosed in US Patent No. 6,492,332 to Demopulos.

The MASP-2 inhibitory agent may be administered to the patient by intraarterial, intravenous, intramuscular, intrathecal, intracranial, subcutaneous or other parenteral administration and potential orally for non-peptidergic inhibitors. Administration may be repeated periodically as determined by a physician for the optimal therapeutic effect. For inhibition of restenosis, MASP-2 inhibitor composition may be administered before and / or durantee / or after placement of a probe or atherectomy or angioplasty procedure. Alternatively, the MASP-2 inhibitor composition may be coated on or incorporated into the probe.

GASTRINTESTINAL DISORDERS

Ulcerative colitis and Crohn's disease are chronic inflammatory bowel disorders that fall under the banner of inflammatory bowel disease (IBD). IBD is characterized by spontaneously occurring, chronic, recurrent inflammation of unknown origin. Despite extensive research into the disease in both human and experimental humans, the precise mechanisms of pathology remain to be elucidated. However, the complement system is believed to be activated in patients with IBD and is considered to play a role in the disease's pathogenesis (Kolios, G., et al., Hepato-Gastroenterology 45: 1601-9, 1998; Elmgreen, J., Dan. Med. Bull. 33: 222, 1986).

C3b and other complement-activated products have been shown to be found on the luminal surface of surface epithelial cells, as well as in IBD-compliant muscle mucosa and submucosal blood vessels (Halstensen, TS, et al., Immunol. Res. 10: 485-92, Halstensen, TS, et al., Gastroenterology 98: 1264, 1990). In addition, polymorphonuclear cell infiltration, usually a result of the generation of C5a, is characteristically observed in the inflammatory bowel (Kohl, J., Mol.Immunol. 38: 175, 2001). The multifunctional complement inhibitor K-76 has also been reported to produce symptomatic improvement of ulcerative colitis in a small clinical study (Kitano, A., et al., Dis. Colon Rectum 35: 560, 1992), as well as in a colitis model. induced carrageenan in rabbits (Kitano, A., et al., Clin. Exp. Immunol. 94: 348-53, 1993).

A novel human C5a receptor antagonist has been shown to protect against disease pathology in a mouse model of EBD (Woodruff, T.M., et al., J. Immunol. 171: 5514-20, 2003). Mice that were genetically deficient in Decline Accelerator Factor (DAF), a membrane complement regulatory protein, were used in an IBD model to demonstrate that DAF deficiency resulted in markedly greater tissue damage and increased proinflammatory cytokine production (Lin , F., et al., J. Immunol. 172: 3836-41, 2004). Therefore, complement control is important in regulating intestinal homeostasis and may be a major pathogenic mechanism involved in the development of IBD.

The present invention thus provides methods for inhibiting MASP-2-dependent complement activation in patients suffering from inflammatory gastrointestinal disorders, including but not limited to pancreatitis, diverticulitis and intestinal disorders including Crohn's disease, ulcerative colitis and irritable bowel syndrome, by the administration of a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier to a patient suffering from such a disorder. The MASP-2 inhibitory agent may be administered to the patient by intraarterial, intravenous, intramuscular, subcutaneous, intrathecal, intracranial or other parenteral administration and potential orally for non-peptidergic inhibitors. Administration may be appropriately repeated periodically as determined by a physician to control symptoms of the disorders that are treated.

PULMONARY CONDITIONS

Complement has been implicated in the pathogenesis of many pulmonary inflammatory disorders, including: acute respiratory distress syndrome (ARDS) (Ware, I., et al., N. Engl. J. Med. 342: 1334-49,2000); transfusion-related acute lung injury (TRALI) (Seeger, W., et al., Blood 76: 1438-44, 1990); acute lung injury deischemia / reperfusion (Xiao, F., et al., J. Appl. Physiol. 82: 1459-65, 1997); chronic obstructive pulmonary disease (COPD) (Marc, MM, et al., Am. J Respirate Cell Mol. Biol (Epub ahead of print), March 23, 2004) Asthma (Krug, N., et al., Am. J. Respir. Crit Care Care Med. 164: 1841-43 , 2001); Wegener's granulomatosis (Kalluri, R., et al., J. Am. Soc. Nephrol. 8: 1795-800, 1997); and antiglomerular-based membrane disease (Goodpasture's disease) (Kondo, C., et al., Clin. Exp. Immunol. 124: 323-9, 2001).

And now it is well accepted that much of the pathophysiology of

ARDS involves a deregulated inflammatory cascade that begins as a normal response to an infection or other inciting event, but ultimately causes significant host injury (Stanley, T. P., Emerging Therapeutic Targets 2: 1-16, 1998). Nearly universal ARDS patients show evidence of extensive complement activation (increased plasma levels of complement components C3a and C5a) and the degree of complement activation was correlated with ARDS development and consequence (Hammerschmidt, DF, et al., Lancet 1: 947-49, 1980; Solomkin, JS, et al., J. Surgery 97: 668-78, 1985). Several experimental and clinical data suggest a role for complement activation in the pathophysiology of ARDS. In deanimal models, systemic complement activation leads to acute lung injury with histopathology similar to that observed in human ARDS (Till, GO, et al., Am. J. Pathol. 129: 44-53, 1987; Ward, A.. A. , Am. J. Pathol. 149: 1081-86, 1996). Inhibiting the complement cascade by general-depletion depletion or by specific inhibition of C5a confers protection in animal models of acute lung injury (Mulligan, M.S., et al., J. Clin.Invest. 98: 503-512, 1996). In rat models, sCR1 has a protective effect on complement and neutrophil-mediated lung injury (Mulligan, M.S., Yeh, et al., J. Immunol. 148: 1479-85, 1992). In addition, virtually all complement components can be locally produced in the lung by type II alveolar cells, alveolar macrophages, and fibroblastospulmonary cells (Hetland, G., et al., Scand. J. Immunol. 24: 603-8, 1986; Rothman, Β I., et al., J. Immunol. 145: 592-98, 1990). Thus, the decomposition cascade is well positioned to contribute significantly to pulmonary inflammation and, consequently, to lung injury in ARDS.

Asthma is, in essence, an inflammatory disease. Cardinal features of allergic asthma include airway hyperresponsiveness to a variety of specific and non-specific stimuli, excessive airway mucus production, pulmonary eosinophilia, and elevated IgE concentration. Although asthma is multifactorial in origin, it is generally accepted that asthma arises as a result of inadequate immune responses to environmental antigens common in genetically susceptible individuals. The fact that the complement system is highly activated in the human asthmatic lung is well documented (Humbles, A., et al., Nature 406: 998-01,2002; van de Graf, A., et al. , J. Immunol Methods 147: 241-50, 1992) In addition, recent data from animal and human models provide evidence that complement activation is an important mechanism contributing to the pathogenesis of the disease (Karp, CL, et al. Nat. Immunol.1: 221-26, 2000; Bautsch, W., et al., J. Immunol. 165: 5401-5, 2000; Drouin, SM, et al., J. Immunol. 169: 5926 -33, 2002; Walters, DM, et al., Am. J. Respir. Cell Mol. Biol. 27: 413-18, 2002). A role for the dalectin pathway in asthma is supported by studies using a murine model of chronic fungal deasma. Mice with a genetic deficiency in lectin that binds manna develop altered airway hyperresponsiveness compared to normal animals in this model of asthma (Hogaboam, C.M., et al., J. Leukoc. Biol. 75: 805-14, 2004).

Complement may be activated in asthma by a variety of pathways, including: (a) activation via the classical pathway as a result of allergen-antibody complex formation; (b) alternate pathway activation on allergenic surfaces; (c) activation of the dalectin pathway through the intermingling of carbohydrate structures in allergens; and (d) cleavage of C3 and C5 by the released proteases of inflammatory cells. While much remains to be learned about the complex role played by complement in asthma, identifying activation of complement pathways involved in the development of allergic asthma may provide a focus for development of new therapeutic strategies for this increasingly important disease.

Several studies using animal models have shown a critical role for C3 and its cleavage product, C3a, in the development of the allergic phenotype. Drouin and colleagues used C3 deficient mice in the ovalbumin (OVA) / Aspergillusfumigatus asthma model (Drouin et al., J. Immunol. 167: 4141-45, 2001). They found that when inoculated with allergen, C3-deficient mice show surprisingly diminished AHR and pulmonary eosinophilia compared to paired wild-type control mice.

In addition, these C3-deficient mice had dramatically reduced numbers of IL-4-producing cells and attenuated Ag-specific IgE and IgG responses. Taube and colleagues obtained similar results in the OVA model of asthma by blocking C3- and C4-level complement-activation activation using a recombinant soluble Crry mouse complement receptor form (Taube et al., Am. J. Respir. Care Med. 168 : 1333-41, 2003). Humbles and colleagues deleted C3aR in mice to examine the role of C3a in eosinophil function (Humbles et al., Nature 406: 998-1001, 2000). Using the OVA deasma model, they observed almost complete protection from the development of AHR for aerosolized methacholine. Drouin and colleagues (2002) used C3aR deficient mice in the OVAJA asthma model. fumigatuse demonstrated an attenuated allergic response very similar to C3-deficient animals with decreased AHR, eosinophilic recruitment, TH2 cytokine production, and lung mucus secretion, as well as reduced Ag-specific IgE and IgG1 responses (Drouin et al., J. Immunol. 169: 5926-33,2002). Bautsch and colleagues conducted investigations using a guinea pig strain that have a natural deletion of C3aR (Bautsch et al., J. Immunol. 165: 5401-05, 2000). Using an OVA model of allergic asthma, they observed significant protection from airway bronchoconstriction following antigen inoculation.

Several recent studies using animal models have shown a critical role for C5 and its C5a cleavage product, the development of the allergic phenotype. Abe and colleagues reported evidence linking C5aR activation with airway inflammation, decytocin production, and airway responsiveness (Abe et al., J. Immunol. 167: 4651-60, 2001). In their studies, inhibition of complement activation by soluble CRls, futhan (a complement activation inhibitor) or synthetic hexapeptide C5a antagonist blocked the inflammatory response and airway responsiveness to methacholine. In studies using an anti-C5 anti-C5 blocker Peng and colleagues found that C5 activation contributed substantially to both airway inflammation and AHR in the OVA model of asthma (Peng et al., J. Clin. Invest. 115: 1590-1600, 2005). Also, Baelder and colleagues reported that C5aR blockade substantially reduced AHR in the asthma model A. fumigatus (Baelder etal., J. Immunol. 174: 783-89, 2005). In addition, blockade of both C3aR and C5aR significantly reduced airway inflammation as shown by the reduced numbers of neutrophils and eosinophils in BAL.

Although the previously listed studies highlighted the importance of complement factors C3 and C5 and their cleavage products in the pathogenesis of experimental allergic asthma, these studies provide no information about the contribution of each of the three complement activation pathways since C3 and C5 are common. to all three activation paths. However, a recent study by Hogaboam and colleagues indicates that the lectin pathway may play a major role in the pathogenesis of asthma (Hogaboam et al., J. Leukocytye Biol. 75: 805-814, 2004). These studies used genetically deficient mice that bind lectin-A (MBL-A), a carbohydrate-binding protein that functions as the recognition component for activation of the lectin-depleting pathway. In a chronic fungal asthma model, A. fumigatus MBL-A (+ / +) and MBL-A (- / -) sensitized mice were examined on days 4 and 28 after an i.t. with A. fumigatus conidia. AHR in sensitized MBL-A (- / -) mice was significantly attenuated both times after inoculation with conidia compared with the sensitized MBL-A (+ / +) group. They found that pulmonary TH2 cytokine levels (IL-4, IL-5 and IL-13) were significantly lower in A. fumigatus-sensitized MBL-A (- / -) mice compared with the wild type group on day 4 afterwards. of the conidia. Their results indicate that MBL-A and the lectin pathway play a major role in the development and maintenance of AHR during chronic fungal asthma.

Results from a recent clinical study in which the association between a specific MBL polymorphism and the development of the asthma provide further evidence that the lectin pathway may play an important pathological role in this disease (Kaur et al., Clin.Experimental Immunol. 143: 414 -19, 2006). Plasma concentrations of MBL vary widely between individuals and this is primarily attributable to genetic polymorphisms within the MBL gene. They found that individuals who carry at least one copy of a specific MBL polymorphism that above regulates MBL expression two to four times have an almost five-fold increased risk of developing bronchial asthma. There was also an increased severity of disease markers in patients with bronchial asthma carrying this MBL polymorphism.

One aspect of the invention thus provides a method for treating pulmonary disorders by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier to a patient suffering from pulmonary disorders, including without limitation acute respiratory distress syndrome. , transfusion-related acute lung injury, acute ischemia / reperfusion lung injury, chronic obstructive pulmonary disease, asthma, Wegener's granulomatosis, baseantiglomerular membrane disease (Goodpasture's disease), meconium aspiration syndrome, bronchiolitis obliterative syndrome, pulmonary fibrosis idiopathic disease, acute pulmonary injury secondary to burn, non-cardiogenic pulmonary edema, transfusion-related respiratory depression and emphysema. The MASP-2 inhibitor may be administered to the patient systemically, such as by intraarterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral administration or potentially by oral administration to non-peptidergic agents. The composition of the MASP-2 inhibitor may be combined with one or more additional therapeutic agents, including anti-inflammatory agents, antihistamines, corticosteroids or antimicrobial agents. Administration may be repeated as determined by a physician until the condition has been resolved.

EXTRACORPORAL CIRCULATION

There are numerous medical procedures during which blood is diverted from a patient's circulatory system (extracorporeal circulation systems or ECC). Such procedures include hemodialysis, plasmapheresis, leukopheresis, extracorporeal membrane oxygenator (ECMO), LDL precipitation on heparin-induced extracorporeal membrane oxygenation (HELP), and cardiopulmonary bypass (CPB). These procedures expose the blood or blood products to foreign surfaces that may alter normal cell function and hemostasis. In pioneering studies Craddocket al. identified complement activation as the probable cause of degranulocytopenia during hemodialysis (Craddock, P. R, et al., N. Engl. J. Med. 296: 769-74, 1977). Results from numerous studies between 1977 and the present indicate that many of the adverse events experienced by patients undergoing hemodialysis or CPB are caused by activation of the complement system (Chenoweth, DE, Ann. NY Acad.Sei. 516: 306-313, 1987). Hugli, TE, Complement 3: 111-127, 1986; Cheung, AK, J. Am. Soc. Nephrol. 1: 150-161, 1990; Johnson, RJ, Nephrol. Dial. Transplant 9: 36-45 1994) . For example, the potential complement activator has been shown to be an important criterion in determining the compatibility of hemodialysis with respect to recovery from renal function, susceptibility to infection, pulmonary dysfunction, and morbidity and survival rate of patients with renal failure (Hakim, RM, Kidney Int. 44 : 484-4946, 1993).

It has been widely believed that hemodialysis membrane activation-decomposition occurs through alternative pathway mechanisms due to weak C4a generation (Kirklin, JK, et al., J. Thorac. Cardiovasc. Surg. 86: 845-57, 1983; Vallhonrat, H ., et al., ASAIO J.45: 113-4, 1999), but recent work suggests that the classical path may also be involved (Wachtfogel, YT, et al., Blood 73: 468-471, 1989).

However, there is still inadequate understanding of the factors that initiate and control complement activation on artificial surfaces including biomedical polymers. For example, a Cuprophan membrane used in hemodialysis has been classified as a highly potent complement activator. While not wishing to be bound by theory, inventors argue that this may be partly explained by its polysaccharide nature. Activation of the MASP-2 dependent complement system identified in this patent provides a mechanism by which activation of the lectin pathway triggers activation of the alternative pathway.

Patients who undergo ECC during CPB suffer from a systemic inflammatory reaction, which is partly caused by exposure of blood to artificial surfaces of the extracorporeal circuit, but also by independent surface factors such as surgical trauma and reperfusion injury (Butler, J., et al. , Ann. Thorac. Surg. 55: 552-9, 1993; Edmunds, LH, Ann. Thorac. Surg. 66 (Suppl): S12-6, 1998; Asimakopoulos, G., Perfusion 14: 269-77, 1999) . The inflammatory reaction triggered by CPB may result in postoperative complications, commonly referred to as "post perfusion syndrome". Among these events are cognitive deficits (Fitch, J., et al., Circulation 100 (25): 2499-2506, 1999), respiratory failure, bleeding disorders, renal dysfunction, and, in the most severe cases, multiple organ failure (Wan, S., et al., Chest 112: 676-692,1997). Coronary bypass surgery with CPB leads to profound decomposition activation, unlike surgery without CPB but with a comparable degree of surgical trauma (E. Fosse, 1987). Therefore, the primary suspected cause of these CPB-related problems is inadequate activation of complement during the shunt procedure (Chenoweth, K., et al., N.Engl. J. Med. 304: 497-503, 1981; P. Haslam, et al., Anesthesia 25: 22-26,1980; J. Kirklin et al., J. Thorac Cardiovasc Surg 86: 845-857 1983; Moore FD et al. Ann Surg 208: 95 (103, 1988; J. Steinberg, et al., J. Thorac. Cardiovasc. Surg 106: 1901-1918, 1993). In CPB circuits, the alternative complement pathway plays a predominant role in complement activation, which results from the interaction of blood with artificial surfaces of the CPB circuits (Kirklin, JK, et al., J. Thorac. Cardiovasc. Surg., 86: 845- 57, 1983; Kirklin, JK, et al., Ann. Thorac. Surg.41: 193-199, 1986; Vallhonrat H., et al., ASAIO J. 45: 113-4, 1999). evidence that the classical complement pathway is activated during CPB (Wachtfogel, YT, et al., Blood 73: 468-471, 1989).

Primary inflammatory substances are generated after activation of the complement system, including anaphylatoxins C3a and C5a, aopsonin C3b and membrane attack complex C5b-9. C3a and C5a are potent stimulators of neutrophils, monocytes and platelets (Haeffner-Cavaillon, N., et al., J. Immunol., 139: 794-9, 1987; Fletcher, MP, et al., Am. J. Physiol. 265: H1750-61, 1993; Rinder, CS, et al., J. Clin Invest 96: 1564-72, 1995; Rinder, CS, et al., Circulation 100: 553-8, 1999). Activation of these cells results in the release of proinflammatory cytokines (IL-1, IL-6, IL-8, TNF alpha), oxidative free radicals, and proteases (Schindler, R., et al., Blood 76: 1631-8, 1990. ; Cruickshank, AM, et al., Clin Sci. (Lund) 79: 161-5, 1990; Kawamura, T., et al., Can. J. Anaesth. 40: 1016-21, 1993; Steinberg, JB, et al., J. Thorac, Cardiovasc. Surg. 106: 1008-1, 1993; Finn, A., et al., J. Thorac. Cardiovasc. Surg. 105: 234-41, 1993; Ashraf, SS, et. al., J. Cardiothorac Vasc Anesth 11: 718-22, 1997). C5a has been shown to overregulate Mac-I CD11b and CD18 adhesion molecules in polymorphonuclear cells (PMNs) and induce degranulation of PMNs to proinflammatory release enzymes. Rinder, C., et al., Cardiovasc Pharmacol. 27 (Suppl1): 56-12, 1996; Evangelist, V., et al., Blood 93: 876-85, 1999; Kinkade, J.M., Jr., et al., Biochem. Biophys. Res. Commun. 114: 296-303, 1983; Lamb, N. J., et al., Crit. Care Med. 27: 1738-44, 1999; Fujie, K., et al., Eur. J.

Pharmacol. 374: 117-25, 1999. C5b-9 induces expression of the P-selectin (CD62P) death molecule on platelets (Rinder, CS, et al., J. Thorac.Cardiovasc. Surg. 118: 460-6, 1999 ), whereas both C5a and C5b-9 induce surface expression of P-selectin in endothelial cells (Foreman, KE, et al., J. Clin. Invest. 94: 1147-55, 1994). These death molecules are involved in the interaction between leukocytes, platelets and endothelial cells. Expression of adhesion molecules in endothelial-activated cells is responsible for sequestering activated leukocytes, which then mediate inflammation and tissue damage (Evangelista, V., Blood 1999; Foreman, KE, J. Clin. Invest. 1994; Lentsch, AB, et al., J. Pathol. 190: 343-8, 2000). It is the actions of these complement activation products in the neutrophils, monocytes, platelets, and other circulatory cells that are likely to lead to the various problems that arise after CPB.

Several complement inhibitors are being studied for potential applications in CPB. These include a recombinant soluble complement receptor 1 (sCR1) (Chai, PJ, et al., Circulation 101: 541-6,2000), a humanized anti-C5 single chain antibody (h5G1.-scFv orPexelizumab) (Fitch , JCK, et al., Circulation 100: 3499-506, 1999), a recombinant human membrane cofactor protein fusion hybrid (CAB-2) and human decline accelerating factor (Rinder, CS, et al., Circulation 100: 553-8, 1999), a C3-binding cyclic peptide residue 13 (Compstatin) (Nilsson, B., et al., Blood 92: 1661-7, 1998) and a DMoAb anti-factor (Fung, M., et al., J. Thoracic Cardiovasc (Surg. 122: 113-22, 2001) .SCR1 and CAB-2 inhibit alternative classical and complement pathways at activation stages C3 and C5. Compstatin inhibits both complement paths in activation step C3, whereas h5Gl.l-scFv does only in activation step C5. MoAb Anti-Factor D inhibits the alternate path in activation steps C3 and C5. However, none of these complement inhibitors can specifically inhibit activation of the MASP-2 dependent complement system identified in this patent.

Results of a large prospective phase 3 clinical study to investigate the efficacy and safety of humanized single chain anti-C5 antibody (h5Gl.l-scFv, pexelizumab) in reducing perioperative MI and mortality in coronary artery bypass graft (CABG) surgery reported (Verrier, ED, et al., JAMA 291: 2319-27, 2004). Compared to complacency, pexelizumab was not associated with a significant reduction in death composite endpoint or MI in 2746 patients who underwent CABG surgery. However, there was a statistically significant reduction 30 days after the procedure among all 3099 patients undergoing CABG surgery with or without valve surgery. Since pexelizumab inhibits the C5 activation step, it inhibits the generation of C5a and sC5b-9 but has no effect on the generation of two other potent complement inflammatory substances, C3a and C3bopsonic, which are also known to contribute to the triggered inflammatory reaction. by the CPB.

One aspect of the invention is thus directed to preventing further inflammatory reaction triggered by extracorporeal exposure by treating a patient undergoing an extracorporeal circulation procedure with a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical vehicle, including patients. undergoing hemodialysis, plasmapheresis, leukopheresis, extracorporeal membrane oxygenation (ECMO), LDL precipitation on heparin-induced extracorporeal membrane oxygenation (HELP), and cardiopulmonary bypass (CPB). Co-efficient treatment of MASP-2 inhibitor according to the methods of the present invention is believed to be useful in reducing or preventing the cognitive dysfunction that sometimes results from CPB procedures. The MASP-2 inhibitory agent may be administered to the patient preprocedural and / or intraprocedural and / or postprocedural, such as intraarterial, intravenous, intramuscular, subcutaneous or other parenteral administration. Alternatively, the MASP-2 inhibitory agent may be introduced into the patient's bloodstream during extracorporeal circulation, such as by injecting the MASP-2 inhibitor into the tubing or a membrane through which or before blood is circulated or by contacting blood with a surface that has been coated with the MASP-2 inhibitory agent such as an inner wall of the pipe, membrane or other surface such as a CPB device.

INFLAMMATORY AND NON-INFLAMMATORY ARTHRITIS AND OTHER ESOUELETAL MUSCLE DISEASES

Activation of the complement system has been implicated in the pathogenesis of a wide variety of rheumatologic diseases; including rheumatoid arthritis (Linton, SM, et al., Molec. Immunol. 36: 905-14, 1999), juvenile rheumatoid arthritis (Mollnes, TE, et al., Arthritis Rheum. 29: 1359-64, 1986), osteoarthritis (Kemp, A.A., et al., J. Clin. Lab. Immunol. 37: 147-62,1992), systemic lupus erythematosus (SLE) (Molina, H., Current Opinion in Rheumatol. 14: 492-497 , 2002), Behcefs syndrome (Rumfeld, W.R. etal., Br. J. Rheumatol. 25: 266-70, 1986) and Sjogren's syndrome (Sanders, ME, etal., J. Immunol. 138: 2095 -9, 1987).

There is compelling evidence that immune-triggered decomposition activation is a major pathological mechanism that contributes to tissue damage in rheumatoid arthritis (RA). There are numerous publications documenting that complement-activation products are elevated in the plasma of RA patients (Morgan, P., et al., Clin. Exp. Immunol, 73: 473-478, 1988; Auda, G., et. al., Rheumatol Int. 10: 185-189, 1990; Rumfeld, WR, et al., Br. J. Rheumatol.25: 266-270 (1986). Complement activation products such as C3a, C5a and sC5b-9 were also found within inflamed rheumatic joints and positive correlations were established between the degree of complement activation and the severity of RA (Makinde, VA, et al., Ann. Rheum Dis 48: 302-306, 1989; Brodeur, JP, et al., Arthritis Rheumatism34: 1531-1537, 1991). In both adult and juvenile rheumatoid arthritis, elevated serum and synovial fluid levels of alternative pathway activation product Bb compared with C4d (a marker for classic pathway activation) indicate that complement activation is predominantly mediated by the alternative pathway ( El-Ghobarey, AFet al., J. Rheumatology 7: 453-460 (1980; Agarwal, A., et al., Rheumatology39: 189-192, 2000). Complement activation products can directly damage tissue (via C5b-9) or indirectly mediate inflammation through the recruitment of inflammatory cells by C3a and C5a anaphylactoxins.

Animal models of experimental arthritis have been widely used to investigate the role of complement in the pathogenesis of ARD. Complement depletion by snake venom factor in RA animal models prevents the onset of arthritis (Morgan, K., et al., Arthritis Rheumat. 24: 1356-1362, 1981; Van Lent, PL, et al., Am. J. Pathol 140: 1451-1461, 1992). Intra-articular injection of the complement receptor 1 soluble form (sCR1), a complement inhibitor, suppressed inflammation in a rat model of RA (Goodfellow, RM, et al., Clin.Exp. Immunol. 110: 45- 52, 1997). In addition, sCR1 inhibits the development and progression of collagen-induced rat arthritis (Goodfellow, R. M., et al., Clin Exp. Immunol. 119: 210-216, 2000). Soluble CR1 inhibits the alternate classical and complement pathways in the C3 and C5 activation steps in both the alternate and classical pathways, thereby inhibiting the generation of C3a, C5a, and sC5b-9 in a timely manner.

In the 1970s it was recognized that immunization of type II heterologous collagen (IIC; the major collagen component of human joint cartilage) leads to the development of autoimmune arthritis (collagen-induced arthritis or ASD) with significant similarities to human RA. (Courtenay, JS, et al., Nature 283: 666-68 (1980), Banda et al., J. of Immunol. 171: 2109-2115 (2003)). Autoimmune response in susceptible animals involves a complex combination of factors including major histocompatibility complex (MHC) molecules, cytokines, and CII-specific BeT cell responses (reviewed by Myers, LK, et al., Life Sciences 61: 1861-78, 1997). The observation that nearly 40% of congenital mouse strains have a complete deficiency in complement component C5 (Cinader, B., et al., J. Exp. Med. 120: 897-902, 1964) provided an indirect opportunity to explore the role. of complement in this arthritic model by comparing deficient and sufficient CIA The results of such studies indicate that sufficiency in C5 is an absolute requirement for the development of ASD (Watson et al., 1987; Wang, Y., et al., J. Immunol. 164: 4340-4347, 2000). Further evidence of the importance of C5 and complement in AR was provided by the use of monoclonal anti-C5 antibodies (MoAbs). Prophylactic intraperitoneal administration of anti-C5 MoAbs in a CIA modelomurine almost completely prevented disease onset while treatment during active arthritis resulted in both clinical benefit and significant milder histological disease (Wang, Y., et al., Proc.Natl Acad Sci USA 92: 8955-59 (1995).

Further insights into the potential role of complement activation in disease pathogenesis have been provided by studies using transgenic TK / BxN cell receptor mice, a newly developed model of inflammatory arthritis (Korganow, AS, et al., Immunity 10: 451-461, 1999). ). All KTBx animals spontaneously developed an autoimmune disease with most (but not all) of the clinical, histological and immunological features of RA in humans. In addition, serum transfer of arthritic K / BxN mice in healthy animals causes arthritis within days through the transfer of arthritogenic immunoglobulins. To identify the specific complement activation steps required for disease development, arthritic K / BxN mouse serum was transferred into several genetically deficient mice for a particular complement pathway product (Ji, H., et al., Immunity 16: 157- 68, 2002). Interestingly, the study results demonstrated that alternate path activation is critical, whereas classic path activation is unnecessary. In addition, C5a generation is critical because both C5-deficient and C5aR-deficient mice were protected from disease development. Compatible with these results, a previous study reported that genetic removal of C5a receptor expression protects mice from arthritis ( Grant, EP, etal., J. Exp. Med. 196: 1461-1471, 2002).

A humanized anti-C5 MoAb (5G1.1) that prevents cleavage of the human complement component C5 into its proinflammatory components is under development by Alexion Pharmaceuticals, Inc., New Haven, Connecticut, as a potential treatment for RA.

Two research groups independently proposed that the lectin pathway promotes inflammation in RA patients through the interaction of MBL with specific IgG glycoforms (Malhotra et al., Nat.Med. 1: 237-243, 1995; Cuchacovich et al., J Rheumatol 23: 44-51, 1996). ARA is associated with a marked increase in galactose-depleting IgG glycoforms (alluded to as IgGO glycoforms) in the Fc damolecule region (Rudd et al., Trends Biotechnology 22: 524-30, 2004). The percentage of IgGO glycoforms increases with progression of erectile disease to normal when patients are in remission. In vivo, IgGO is deposited in synovial tissue and MBL is present at increased levels in synovial fluid in individuals with RA. Aggregated agalactosyl IgG (IgGO) clustered IgG associated with RA can bind mannose binding lectin (MBL) and activate the complement lectin pathway. In addition, results from a recent clinical study examining allelic variants of MBL in RA patients suggest that MBL may have an enhancing role in inflammatory disease (Garred et al., J. Rheumatol. 27: 26-34, 2000). Therefore, lectin pathogen may play an important role in the pathogenesis of RA.

Systemic lupus erythematosus (SLE) is an autoimmune disease of undefined etiology that results in the production of autoantibodies, generation of circulatory and episodic immune complexes, uncontrolled activation of the complement system. Although the origins of autoimmunity in SLE remain difficult to understand, considerable information is now available implying complement activation as an important mechanism contributing to vascular injury in this disease (Abramson, SB, et al., Hospital Practice 33: 107-122, 1998). . Activation of both classical and alternative pathways of decomposition is involved in disease both C4d and Bb are sensitive markers of moderate to severe delupus disease activity (Manzi, S., et al., Arthrit. Rheumat. 39: 1178-1188, 1996 ). Activation of the alternative complement pathway accompanies disease outbreaks in systemic lupus erythematosus during pregnancy (Buyon, J. P., et al., Arthritis Rheum. 35: 55-61, 1992). In addition, the lectin pathway may contribute to the development of the disease because autoantibodies against MBL have recently been identified in our patients from SLE patients (Seelen, A., et al., Clin Exp. Immunol. / 34: 335-343, 2003 ). The immune-mediated complex by decomposition activation via the classical pathway is believed to be a mechanism by which tissue alleviation occurs in SLE patients. However, inherited deficiencies in classic pathway complement components increase the risk of lupus and lupus-equivalent disease (Pickering, M.C., et al., Adv.Immunol. 76: 227-324, 2000). SLE or a related syndrome occurs in more than 80% of people with complete deficiency of Clq, Clr / Cls, C4or C3. This presents an obvious paradox in the reconciliation of harmful effects with complement protective effects in lupus.

An important activity of the classical pathway seems to be the promotion of the removal of immune complexes from the circulation and tissues by the mononuclear phagocytic system (Kohler, P. F., et al., Am. J. Med. 56: 406-11,1974). In addition, complement has recently been found to play an important role in the removal and disposal of apoptotic bodies (Mevorarch, D., etal., J. Exp. Med. 188: 2313-2320, 1998). Deficiencies in classic pathway function may predispose patients to SLE development by allowing a developmental cycle in which complexes or immune cells apoptotic cells accumulate in tissues, causing inflammation and autoantigen release, which in turn stimulates the production of autoantibodies and more immune complexes. and thus evoke an autoimmune response (Botto, M., et al., Nat. Genet. 19: 56-59, 1998; Botto, M., Arthritis Res. 3: 201-10, 2001). However, these "complete" deficiency states in the classical pathway components are present in approximately one of 100 SLE patients. Therefore, in the vast majority of SLE patients, complement deficiency in the classic pathway components does not contribute to the etiology of the disease and complement activation may be an important mechanism contributing to the pathogenesis of SLE. The fact that many individuals with permanent genetic deficiencies in the classic pathway components often develop SLE at some point in their lives proves the redundancy of mechanisms capable of triggering the disease.

Results from animal models of SLE support the important role of complement activation in disease pathogenesis. Inhibiting C5 activation using an anti-C5 blocking MoAb decreased proteinuria and kidney disease in NZB / NZW F1 mice, an SLE mouse model (Wang Y., et al., Proc. Natl. Acad. Sci. USA 93: 8563- 8, 1996). In addition, anti-C5 MoAb treatment of mice with severe combined immune deficiency disease implanted with cells secreting anti-DNA antibodies results in improved proteinuria and renal histological picture with an associated benefit in survival compared with untreated controls (Ravirajan, CT, et al. al., Rheumatology 43: 442-7,2004). The alternative pathway also plays an important role in the manifestations of SLE autoimmune disease as the backcrossing of factor B deficient mice in the SLE MRL / lpr model revealed that lack of factor B decreased vasculitis, glomerular disease, C3 consumption, and blood levels. IgG3 RF typically found in this model without altering levels of other autoantibodies (Watanabe, H., et al., J. Immunol. 164: 786-794, 2000). A humanized anti-C5 MoAb is under investigation as a potential treatment for SLE. This antibody prevents cleavage from C5 to C5a and C5b. In Phase I clinical trials, no serious adverse effects were observed and more human trials are underway to determine efficacy in SLE (Strand, V., Lupus 10: 216-221, 2001).

Results from both human and deanimal studies support the possibility that the complement system directly contributes to the pathogenesis of muscular dystrophy. Studies of human distrophic biopsies have shown that C3 and C9 are deposited on both necrotic and non-necrotic fibers in the dystrophic muscle (Cornelio eDones, Ann. Neurol. 16: 694-701, 1984; Spuler and Engel, AG, Neurology 50: 41-46. , 1998). Using DNA micro-series methods, Porter and colleagues discovered the markedly enhanced gene expression of numerous complement-related mRNAs in dystrophin-deficient (mdx) mice coincident with the development of dystrophic disease (Porter et al., Hum. Mol. Genet. 11 : 263-72, 2002).

Mutations in the human gene encoding dysferlin, a transmembrane muscle protein, have been identified as major derisis factors for two forms of skeletal muscle disease, namely limb muscular dystrophy (LGMD) and Miyoshi myopathy (Liu et al., Nat. Genet. 20: 31-6, 1998). Several mouse models with dysferlin mutations have been developed and these have also developed progressive muscular dystrophy. Activation of the decompression cascade has been identified on the surface of non-necrotic muscle fibers in some LGMD patients (Spuler and Engel., Neurology 50: 41-46, 1998). In a recent study, Wenzel and colleagues showed that both murine and human dysferlin deficient muscle fibers lack complement inhibitory factor CD33 / DAF, a specific inhibitor of C5b-9 MAC (membrane attack complex) (Wenzel et al. J. Immunol 175: 6219-25, 2005). As a consequence, dysferlin deficient non-necrotic muscle cells are more susceptible to complement-mediated cell lysis. Wenzel and colleagues suggest that complementation of skeletal muscle cells may be a major pathological mechanism involved in the development of LGMD and Miyoshi disease in patients. Connolly and colleagues studied the role of C3 complement in the pathogenesis of a severe model of congenital dystrophy, the dy - / - mouse, which is deficient in laminin a2 (Connolly et al., J. Neuroimmunol. 127: 80-7, 2002). They generated genetically efficient animals in both C3 and laminin a2 and found that the absence of C3 prolonged survival in the dy - / - muscular dystrophy model. In addition, double dead mice (C3 - / -, dy - / -) showed more muscle strength than dy - / - mice. This work suggests that the complement system may contribute directly to the pathogenesis of this congenital dystrophy form.

One aspect of the invention is thus directed to the prevention or treatment of inflammatory and noninflammatory atritis and other skeletal disorders, including but not limited to osteoarthritis, arthritis, juvenile rheumatoid arthritis, gout, neuropathic arthropathy, arthritis psoriatic, ankylosing spondylitis, and other spondyloarthritis, arthropathy muscular dystrophy or systemic lupus erythematosus (SLE) by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical vehicle to a patient suffering from such a disorder. The MASP-2 inhibitory agent may be administered to the patient systemically, such as by intraarterial, intravenous, intramuscular, subcutaneous or other parenteral administration or potentially by oral administration to non-peptideric agents. Alternatively, administration may be by local release, such as by intraarticular injection. The MASP-2 inhibitory agent may be administered periodically over an extended period of time for the treatment or control of a chronic condition or may be administered single or repeatedly prior to, during and / or after acute trauma or injury, including surgical procedures performed. together.

RENAL CONDITIONS

Activation of the complement system has been implicated in the pathogenesis of a wide variety of kidney diseases; including mesangioproliferative glomerulonephritis (IgA nephropathy, Berger disease) (Endo, M., et al., Clin. Nephrology 55: 185-191, 2001), glomerulonephritisemembranosa (Kerjashki, D., Arch B Cell Pathol. 58: 253 -71, 1990; Brenchley, PE, et al., Kidney Int., 41: 933-7, 1992; Salant, DJ, et al., Kidney Int. 35: 976-84, 1989), membranoproliferative glomerulonephritis (mesangiocapillary glomerulonephritis) (Bartlow, BG, et al., Kidney Int. 15: 294-300, 1979; Meri, S., et al., J. Exp. Med. 175: 939-50, 1992), post infection acute glomerulonephritis (glomerulonephritis streptococcal), cryoglobulinemic glomerulonephritis (Ohsawa, I., et al., Clin Immunol. 101: 59-66, 2001), lupus nephritis (Gatenby, A., Autoimmunity 11: 61-6, 1991) and nephritis. Henoch-Schonlein purpura (Endo, M., et al., Am. J. Kidney Dis.35: 401-407, 2000). Complement involvement in kidney disease has been evaluated for several decades but there is still greater debate about its exact role in the early stage, development and resolution of the disease. Under normal conditions the complement contribution is beneficial to the host, but inadequate complement activation and deposition may contribute to tissue injury.

There is substantial evidence that glomerulonephritis, inflammation of the glomeruli, is often initiated by immune complex deposition on the glomerular or tubular structures that then triggers complement activation, inflammation, and tissue injury. Kahn and Sinniah demonstrated increased deposition of C5b-9 on tubular-based membranes in biopsies taken from patients with various forms of glomerulonephritis (Kahn, T.N., et al., Histopath. 26: 351-6, 1995). In a study of patients with IgA nephrology (Alexopoulos, A., et al., Nephrol.Dial. Transplant 10: 1166-1172, 1995), C5b-9 deposition in tubular epithelial / base membrane structures correlated with plasma creatine levels . Another study of membrane nephropathy demonstrated a relationship between clinical outcome and urinary sC5b-9 levels (Kon, S. P., et al., Kidney Int. 48: 1953-58, 1995). Elevated desC5b-9 levels were positively correlated with poor prognosis. Lehto et al. Measured elevated levels of CD59, a regulatory regulatory factor that inhibits membrane attack complex in plasma membranes, as well as C5b-9 in the urine of patients with glomerulonephritis membrane (Lehto, T., et al., Kidney Int. 47: 1403-11, 1995). Histopathological analysis of biopsy specimens taken from these same patients showed deposition of C3 and C9 proteins in the glomeruli, whereas CD59 expression in these tissues was decreased compared with that of normal renal detainees. These various studies suggest that progress of complement-mediated daglomerulonephritis results in urinary excretion of complement proteins that correlate with the degree of injury sustained and disease prognosis.

Inhibition of complement activation in various deanimal models of glomerulonephritis also demonstrated the importance of complement activation in the etiology of the disease. In a model of Membranoproliferative Glomerulonephritis (MPGN), anti-Thil antiserum infusion into C6-deficient mice (which cannot form C5b-9) resulted in 90% less glomerular cell proliferation, 80% reduction in platelet and macrophage infiltration, synthesis. decreased collagen type IV (a marker for mesangial matrix expansion) and 50% less proteinuria than in normal C6 + mice (Brandt, J., et al., Kidney Int. 49: 335-343, 1996). These results implicate C5b-9 as a major mediator of complement tissue damage in this rat anti-thymocyte serum model. In another glomerulonephritis model, infusion of graded dosages of rabbit anti-mouse glomerular base membranes produced a polymorphonuclear leukocyte (PMN) -dependent influx that was attenuated by prior snake venom factor treatment (to consume complement) (Scandrett, AL , et al., Am. J. Physiol. 268: F256-F265,1995). Rats treated with snake venom factor also showed decreased histopathology, decreased long-term proteinuria and lower decreatinin levels than control rats. Using three mouse deGN models (anti-thymocyte serum, Con A anti-Con A, and Heymann passive nephritis), Couser et al. Demonstrated the potential therapeutic efficacy of methods for inhibiting complement by the use of recombinant sCRl protein (Couser, WG, et al. al., J. Am. Soc. Nephrol. 5: 1888-94, 1995). The sCRl-treated mice showed significantly decreased PMN, platelet and macrophage influx, decreased mesangiolysis and proteinuria versus control mice. Further evidence for the importance of glomerulonephritis complement activation was provided by the use of an anti-C5 MoAb in the NZB / W F1 mouse model. Anti-C5 MoAb inhibits C5 cleavage, thereby blocking generation of C5a and C5b-9. Continuous therapy with anti-C5 MoAb for 6 months resulted in significant improvement in the course of daglomerulonephritis, a humanized anti-C5 MoAb monoclonal antibody (5G1.1) that prevents cleavage of human complement component C5 in its proinflammatory components is under development by Alexion Pharmaceuticals, Inc., New Haven, Connecticut, as a potential treatment for glomerulonephritis.

Direct evidence for a pathological role of complement in renal injury is provided by studies of patients with genetic deficiencies in specific complement components. Several reports have documented an association of kidney disease with complement regulatory defactor H deficiencies (Ault, BH, Nephrol. 14: 1045-1053,2000; Levy, M., et al., Kidney Int. 30: 949-56, 1986; Pickering, MC, et al., Nat. Genet. 31: 424-8, 2002). Factor H deficiency results in low factor B and C3 plasma levels and C5b-9 consumption. Both atypical membranoproliferative agglomerulonephritis (MPGN) and idiopathic hemolytic neurological syndrome (HUS) are associated with defactor H deficiency. H-factor deficient pigs (Jansen, JH, et al., Kidney Int. 53: 331-49, 1998) and mice inactivating factor H (Pickering, MC, 2002) show symptoms equivalent to MPGN, confirming the importance of factor H in complement regulation. Deficiencies of other complement components are associated with kidney disease secondary to the development of systemic lupus erythematosus (SLE) (Walport5 MJ, Davies, et al., Ann. Y. Acad. Sci. 815: 267-81, 1997 ). Deficiencies in Clq, C4 and C2 strongly predispose to SLE development through mechanisms that address defective clearance of immune complexes and apoptotic material. In many of these SLE patients lupus nephritis occurs, characterized by the deposition of immune complexes throughout the glomerulus.

Additional evidence linking complement activation and arenal disease was provided by the identification in patients of autoantibodies directed against complement components, some of which were directly related to kidney disease (Trouw, LA, et al., Mol. Immunol. 38: 199-206 , 2001). Several of these autoantibodies show such a high degree of correlation with kidney disease that the term nephritic factor (NeF) was introduced to indicate this activity. In clinical studies, about 50% of nephritic positive patients developed MPGN (Spitzer, RE, et al., Clin. Immunol. Immunopathol. 64: 177-83, 1992) .C3NeF is an autoantibody directed against the alternative pathway daconvertase C3 (C3bBb) and stabilizes this convertase, thereby promoting alternate pathway activation (Daha, MR, et al., J. Immunol. 116: 1-7, 1976). Similarly, autoantibody with a specificity for the classical pathway of C3 convertase (C4b2a), called C4NeF, stabilizes this convertase and thereby promotes classic pathway activation (Daha, MR et al., J. Immunol. 125: 2051-2054, 1980 Halbwachs, L., et al., J. Clin Invest 65: 1249-56 (1980)). The Anti-Clq autoantibodies have been reported to be related to SLE patient nephritis (Hovath, L., et al., Clin. Exp. Rheumatol. 19: 667-72, 2001; Siegert, C., et al., J. Rheumatol 18: 230-34, 1991; Siegert, C., et al., Clin.Exp. Rheumatol. 10: 19-23, 1992) and an increase in the titer of anti-Clq antibody antibodies has been reported to predict a nephritis outbreak ( Coremans, IE, et al., Am. J. Klidney Dis. 26: 595-601, 1995). Immune deposits eluted from postmortem kidneys of SLE patients revealed the accumulation of these anti-Clq autoantibodies (Mannick, M., et al., Arthritis Rheumatol. 40: 1504-11, 1997). All these facts indicate a pathological role for these autoantibodies. However, not all patients with anti-Clq autoantibodies develop kidney disease, and some healthy individuals also have low titers of anti-Clq autoantibodies (Siegert, C.E., et al., Clin. Immunol. Immunopathol. 67: 204-9, 1993).

In addition to the alternative and classical pathways of complement activation, the lectin pathway may also have an important pathological role in kidney disease. Elevated levels of MBL, MBL-associated protein serine, and complement activation products were detected by immunohistochemistry techniques on renal biopsy material from patients diagnosed with several different renal diseases, including Henoch-Schonlein purpura nephritis (Endo, M., et al. J. Amid J. Disney 35: 401-407, 2000), cryoglobulinemic glomerulonephritis (Ohsawa, I., et al., Clin. Immunol. 101: 59-66, 2001) and IgA neuropathy (Endo, M. , et al., Clin Nephrology 55: 185-191, 2001). Therefore, despite the fact that an association between complement and kidney disease has been known for several decades, the data on how complement exactly influences kidney disease is far from complete.

One aspect of the invention is thus directed to the treatment of renal conditions including but not limited to blood-proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis (mesangiocapillary glomerulonephritis), acute infection (post-streptococcal glomerulonephritis, post-streptococcal glomerulonephritis) IgA by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibiting agent in a pharmaceutical carrier to a patient suffering from such a disorder. MASP-2 inhibitor agent may be administered to patients systematically, such as by intraarterial, intravenous, intramuscular, subcutaneous or other parenteral administration or potentially by oral administration to non-peptideric agents. The MASP-2 inhibitor agent may be administered periodically over an extended period of time for the treatment or control of a chronic condition or may be by single or repeated administration in the period before, during or after acute trauma or injury.

SKIN DISORDERS

Psoriasis is a chronic, debilitating skin condition that affects millions of people and is attributed to both genetic and environmental factors. Topical agents such as UVB and PUVA phototherapy are generally considered to be the first line treatment for apsoriasis. However, for generalized or more extensive disease, systemic therapy is indicated as a primary treatment or, in some cases, for potentiation of UVB and PUVA therapy.

The underlying etiology of various skin diseases such as apsoriasis supports a role for immune and proinflammatory processes including involvement of the complement system. In addition, the role of the complement system has been established as an important non-specific skin defense mechanism. Their activation leads to the generation of products that not only help to maintain normal host defenses, but also mediate inflammation and tissue damage. Proinflammatory breakdown products include large opsonic C3 fragments and cell stimulating activities (C3b and C3bi), low-weight pesomolecular anaphylatoxins (C3a, C4a and C5a) and membrane attack complexes. Among them, C5a or its degradation product C5a des Arg appear to be the most important mediators because they exert a potent chemotactic effect on inflammatory cells. Intradermal administration of anaphylatoxin C5 induces skin changes very similar to those observed in cutaneous hypersensitivity vasculitis that occurs through the immune-mediated complex by complement activation. Complement activation is involved in the pathogenesis of inflammatory changes in autoimmune bullous dermatoses. Complement activation by the pemphigus antibody in the epidermis appears to be responsible for the development of the inflammatory changes characteristic of eosinophilic spongiosis. In bullous pemphigoid (BP), interaction of base membrane zone antigen and BP antibody leads to activation of the complement that appears to be related to the leukocytes lining the dermoepidermal junction. The resulting anaphylatoxins not only activate infiltrating leukocytes but also induce mastoid cell degranulation, which facilitates dermoepidermal separation and eosinophil infiltration. Similarly, complement activation appears to play a more direct role in dermoepidermal separation observed in acquired bullous epidermolysis and gestational herpes.

The evidence for complement involvement in psorias comes from recent experimental findings described in the literature related to the pathophysiological mechanisms for inflammatory changes in napsoriasis and related diseases. A growing body of evidence has indicated that T-cell mediated immunity plays an important role in the onset and maintenance of psoriatic lesions. It has been revealed that lymphokines produced by activated T cells in psoriatic lesions have a strong influence on epidermal proliferation. The characteristic neutrophilic accumulation under the stratum corneum can be observed in the highly inflamed areas of psoriatic lesions. Neutrophils are chemotacticattracted and activated by the synergistic action of chemokines, IL-8 and Gro-alpha-liberated by stimulated keratinocytes and particularly by C5a / C5ades-arg produced by activating the alternative complement pathway (Terui, T., Tahoku J. Exp. 190: 239-248, 2000; Terui, T., Exp. Dermatol 9: 1-10, 2000).

Psoriatic scale extracts contain a single fraction of the chemotactic peptide that is likely to be involved in the induction of rhythmic transepidermal leukocyte chemotaxis. Recent studies have identified the presence of two unrelated chemotactic peptides in this fraction, namely C5a / C5a des Arg and interleukin 8 (IL-8) and their related cytokines. To investigate their relative contribution to transsepidermal leukocyte migration as well as their interrelationships in the psoriatic lesions, the immunoreactive C5a / C5a desArg and IL-8 concentrations in the psoriatic lesion scale extracts and those of related sterile pustular dermatoses were quantified. It was found that the concentrations of C5a / C5a desArg and IL-8 were more significantly increased in the lesion skin horny tissue extracts than in those of noninflammatory orthokeratotic skin. The increase in C5a / C5adesArg concentration was specific for lesion scale extracts. Based on these results, it appears that C5a / C5a desArg is generated only in inflammatory lesional skin under specific circumstances that preferentially favors complement activation. This provides a rationale for using a complement activation inhibitor to improve psoriatic lesions.

Although the classical complement pathway has been shown to be activated in psoriasis, there are fewer reports of alternative pathway involvement in inflammatory reactions in psoriasis. Within the conventional perspective of complement pathway activation, complement fragments C4d and Bb are released as activation. of the classical and alternative paths, respectively. The presence of the C4d or Bb fragment therefore denotes a decomposition activation that continues through the classical and / or alternative pathways. One study measured the levels of C4d and Bb in psoriatic scale extracts using enzyme immunoassay techniques. The scales of these dermatoses contained higher levels of C4d and Bb detectable by the enzyme enzyme immunoassay than those in the noninflammatory skin stratum corneum (Takematsu, H., et al., Dermatological 181: 289-292, 1990). These results suggest that the alternative pathway is activated beyond the classical pathway of psoriatic lesional complement.

Additional evidence for the involvement of complement napsoriasis and atopic dermatitis was obtained by measuring normal decomposition components and peripheral blood activation products from 35 patients with atopic dermatitis (AD) and 24 patients with mild to intermediate psoriasis. C3, C4 and Cl inactivator levels (ClINA) were determined in serum by radial immunodiffusion, while C3a and C5a levels were measured by radioimmunoassay. Compared with healthy non-atopic controls, C3, C4 and Cl INA levels were found to be significantly increased in both diseases. In ADD, there was a tendency toward increased C3a levels, whereas in psoriasis, C3a levels were significantly increased. The results indicate that in both AD and psoriasis, the complement system participates in the inflammatory process (Ohkonohchi, K., et al., Dermatological 179: 30-34, 1989).

Complement activation in psoriatic lesion skin also results in deposition of terminal complement complexes within the epidermis as defined by measuring SC5b-9 levels in plasma and corneal tissues of psoriatic patients. SC5b-9 levels in psoriatic plasma were found to be significantly higher than those of controls or those of patients with atopic dermatitis. Studies of total lesion skin protein extracts have shown that while no SC5b-9 can be detected in noninflammatory horny tissues, there have been high levels of SC5b-9 in lesionous horny tissues of psoriasis. By immunofluorescence using a monoclonal antibody to the C5b-9 neoantigen, the deposition of C5b-9 was observed only in the psoriatic skin stratum corneum. In short, in psoriatic lesion skin, the complement system is activated and complement activation proceeds all the way to the terminal stage, generating the membrane attack complex.

New biological drugs that selectively target the immune system have recently become available to treat psoriasis. Four biological drugs that are currently FDA-approved or in Phase 3 studies are: alefacept (Ameviv®) and efalizuMoAb (Raptiva®) which are cell modulators. T; etanercept (Enbrei®), a soluble TNF receptor; and inflixiMoAb (Remicade®) an anti-TNF anti-colon. Raptiva is an immune response modifier, wherein the targeted mechanism of action is a blockade of the interaction between LFA-I in lymphocytes and ICAM-I in antigen presenting cells and vascular endothelial cells. Raptiva binding of CD11a results in available CD11 saturation of a binding site on lymphocytes and infra-modulation of cell surface CD11a expression in lymphocytes. This donor mechanism inhibits T-cell activation, dermal cell trafficking, and T-cell epidermis and reactivation. Thus, a multitude of scientific evidence indicates a role for complement in skin inflammatory disease states and recent pharmaceutical methods have targeted the immune system. or specific inflammatory processes. None, however, identified MASP-2 as a targeted method. Based on the inventors' new understanding of the role of MASP-2 in complement activation, the inventors believe that MASP-2 is an effective target for the treatment of psoriasis and other dermal disorders.

One aspect of the invention is thus directed to the treatment of psoriasis, autoimmune bullous dermatoses, eosinophilic spongiosis, bullous pemphigoid, acquired bullous epidermolysis, atopic dermatitis, herpes management, and other dermal and chemical burn treatment including capillary effusion thereby caused by the administration thereof. A composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical vehicle to a patient suffering from such a skin disorder. The MASP-2 inhibitory agent may be administered to the patient topically by applying a spray, lotion, gel, paste, ointment or irrigation solution containing the MASP-2 inhibitory agent or systemically such as intraarterial, intravenous, intramuscular, subcutaneous administration. or other parenteral administration or potentially by oral administration to non-peptidergic inhibitors. Treatment may involve single administration or repeated dosing or dosing for an acute condition or periodic dosing or dosing to control a chronic condition.

TRANSPLANT

Activation of the complement system significantly contributes to the inflammatory reaction after solid organ transplantation. In transplantation, the complement system can be activated by the blood / reperfusion and possibly by the graft-directed antibodies (Baldwin, W. M., et al., Springer Seminol Immunopathol. 25: 181-197, 2003). In non-primate xenograft for primates, the major complement activators are pre-existing antibodies. Animal model studies have shown that the use of complement inhibitors can significantly prolong graft survival (see below). Thus, there is an established role of the complement system in organ damage after organ transplantation and therefore the inventors believe that I dare to MASP-2 targeted complement inhibitors may prevent graft oluction after allo- or xenotransplantation. Innate immune mechanisms, particularly decomposition, play a larger role in the inflammatory and immune responses against the graft than previously recognized. For example, activation of the alternative complement pathway appears to mediate renal ischemia / reperfusion injury and nearby tubular cells may be both the site and the site of attack of complement components in this setting. Locally produced complement in the kidney also plays a role in the development of both cell and porantibody-mediated immune responses against the graft.

C4d is the degradation product of complement-activated factor C4, a component of the classical and lectin-dependent pathways. C4d targeting has emerged as a useful marker of humoral rejection in both acute and chronic settings and has led to renewed interest in the significance of anti-donor antibody formation. The association between C4d and morphological signs of acute cell rejection is statistically significant. C4d is found in 24 to 43% of Type I episodes, 45% of Type II rejection and 50% of Type III rejection (Nickeleit, V., et al., J. Am.Soe. Nephrol. 13: 242-251, 2002; Nickeleit, V., et al., Nephrol. Dial.Transplant 18: 2232-2239, 2003). Several therapies are under development that inhibit complement or reduce local synthesis as a means to achieve an improved clinical outcome following transplantation.

Activation of the complement cascade occurs as a result of various processes during transplantation. The present therapy, although effective in limiting cell rejection, does not fully address all barriers faced. These include humoral rejection and chronic allograft nephropathy or dysfunction. Although the overall response to the transplanted organ is a result of various effector mechanisms in the host part, complement may play a role in some of these. In the renal transplant scenario, local complement synthesis by nearby cell cells is of particular importance.

The availability of specific complement inhibitors may provide the opportunity for an improved clinical outcome following organ transplantation. Inhibitors that act by a mechanism that blocks complement attack may be particularly useful because they hold the promise of increasing the effectiveness and elimination of systemic complement depletion in an already immune-compromised receptor.

Complement also plays a critical role in xenograft rejection. Therefore, effective complement inhibitors are of great interest as potential therapeutic agents. In pig-to-primate organ transplantation, hyperacute rejection (HAR) results in antibody deposition and complement activation. Strategies and multiple targets were tested to prevent rejection of pig-primate combination hyperagudone xenograft. These methods were performed by removing natural antibodies, complement depletion with snake poison factor, or preventing C3 activation with the soluble decomposition inhibitor sCRl. In addition, complement-activation blocker 2 (CAB-2), a recombinant soluble chimeric protein derived from human decay acceleration factor (DAF) and membrane doco-factor protein, inhibits both C3 and C5 tantoclastic pathway convertses. CAB-2 reduces complement-mediated tissue injury from an ex vivo perfused pig heart with human blood. A study of the efficacy of CAB-2 when a heterotopically transplanted pig heart in rhesus monkeys not receiving any immunosuppression showed that survival of the graft was significantly longer in monkeys receiving CAB-2 (Salerno, CT, et al., Xenotransplantation 9: 125). -134, 2002). CAB-2 markedly inhibited complement activation, as shown by a strong reduction in C3a and SC5b-9. In graft rejection, the tissue deposition deiC3b, C4 and C9 was similar or slightly reduced from that of controls and IgG, IgM5 Clq and fibrin adherence did not change. Thus, this method for complement inhibition nullified the hyperacute rejection of porcine transplanted hearts in rhesus monkeys. These studies demonstrate the beneficial effects of complement inhibition on survival and the inventors believe that inhibition of MASP-2 may also be useful in transplantation.

Another method has focused on the determination of anti-complement 5 (C5) monoclonal antibodies could prevent hyperacute aeration (HAR) in a pre-sensitized mouse rat heart transplant model and whether this MoAb, combined with cyclosporin and cyclophosphamide, could achieve survival graft lengthens. Anti-C5 MoAb has been found to prevent HAR (Wang, H. et al., Transplantation 68: 1643-1651, 1999). The inventors thus believe that other targets in the complement cascade, such as MASP-2, may also be valuable in preventing HAR and acute vascular rejection in future clinical transplantation.

Although the central role of complement in hyperacute rejection observed in xenografts is well established, a subtle role in allogeneic transplantation is emerging. A link between complement and acquired immune response has long been known, with the discovery of complement-depleted quanima mounted subnormalantibody responses following antigen stimulation. Antigen opsonization as a product of complement division C3d has been shown to greatly increase the efficiency of antigen presentation to B cells and has been shown to act by interlocking the type 2 complement receptor on certain B cells. a mouse skin graft model where C3 and C4 deficient mice had a marked defect in allo-antibody production due to insufficient class switching to high affinity IgG. The importance of these mechanisms in kidney transplantation is increased due to the significance of anti-donor antibodies and humoral rejection.

Previous work has shown that C3 synthesis is up-regulated by nearby tubular cells during allograft rejection following renal transplantation. The role of locally synthesized complement was examined in a mouse kidney transplant model. C3-negative donor grafts transplanted into sufficient C3 receptors demonstrated prolonged survival (> 100 days) compared to C3-positive donor control grafts, which were rejected within 14 days. In addition, anti-donor T-cell proliferative response at C3-negative graft recipients was markedly reduced compared to those controls, indicating a locally synthesized C3 effect on T-cell priming.

These observations suggest the possibility that donor antigen exposure to T cells first occurs in the graft and that antigen presentation enhances the locally synthesized complement by donor antigen scavenging or providing additional signals to both antigen and T-cell cells. In the transplanterenal, complement-producing tubular cells also demonstrate complement uptake on their cell surface.

One aspect of the invention is thus directed to preventing further inflammatory reaction resulting from solid organ or tissue transplantation by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibiting agent in a pharmaceutical transplant recipient vehicle, including patients. have received allograft or whole organ xenograft (eg, kidney, heart, liver, pancreas, lung, cornea, etc.) or grafts (eg valves, tendons, bone marrow, etc.). The inhibitor of DEMASP-2 may be administered to the patient by intraarterial, intravenous, intramuscular, subcutaneous or other parenteral administration or potentially by oral administration to non-peptideric inhibitors.

Administration may occur during the acute period following transplantation or as long-term post-transplant therapy. Additionally or instead of post-transplant administration, the patient may be treated with the MASP-2 inhibitor agent prior to transplantation and / or during the transplant procedure, and / or by pretreatment of the organ or tissue to be transplanted with the MASP-2 inhibitor agent. 2. Pretreatment of the organ or tissue may result in the application of a solution, gel or paste containing the MASP-2 inhibitory agent to the surface of the organ or tissue by spraying or irrigating the surface or organ or tissue may be soaked in a solution containing the MASP inhibitor. -2.

CENTRAL AND PERIPHERAL NERVOUS SYSTEM DISORDERS AND INJURIES

Activation of the complement system has been implicated in the pathogenesis of a variety of diseases or injuries of the central nervous system (CNS) or peripheral nervous system (PNS), including but not limited to multiple sclerosis (MS), myasthenia gravis (MG), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Guillain Barre syndrome, subsequent reperfusion of stroke, degenerative discs, brain trauma, Parkinson's disease (PD) and Alzheimer's disease (AD). The initial determination that complement proteins are synthesized in CNS cells including neurons, astrocytes and microglia, as well as the understanding that anaphylatoxins generated in CNS following complement activation may alter neuronal function, clarified the potential role of complement in CNS disorders. (Morgan, BP, et al., ImmunologyToday 17: 10: 461-466, 1996). It has now been shown that C3a receptors and C5a receptors are found in neurons and show widespread distribution in distinct portions of the sensory brain systems, the elbic motors (Barum, S. R., Immunologic Research 26: 7-13, 2002). In addition, anaphylatoxins C5a and C3a have been shown to alter docomer and drinking behavior in rodents and may induce calcium signaling in microglia and neurons. These findings increase the possibilities regarding the therapeutic utility of inhibiting complement activation in a variety of inflammatory CNS diseases including brain trauma, demyelination, meningitis, stroke, and Alzheimer's disease.

Brain trauma or bleeding is a common clinical problem and complement activation may occur and exacerbate resulting in inflammation and edema. Complement inhibition effects were studied in a rat traumatic brain model (Kaczorowski et al., J. Cereb.Blood Flow Metab. 15: 860-864, 1995). Administration of sCRlimitely before brain injury markedly inhibited deneutrophil infiltration into the injured area, indicating that complement was important for phagocytic cell recruitment. Similarly, decomposition activation in patients following cerebral hemorrhage is clearly implicated by the presence of high levels of multiple activation of decomposition products in both plasma and cerebrospinal fluid (CSF). Complement activation and increased dyeing of C5b-9 complexes have been demonstrated in sequestered lumbar disc tissue and may suggest a sciatic role induced by disc herniation tissue (Gronblad, M., et al,

Spine 28 (2): 114-118, 2003).

MS is characterized by a progressive loss of myelin sheath and axon heat stroke within the CNS. Although the initial cause is unknown, there is abundant evidence implicating the immune system (Prineas, JW, et al., Lab Invest. 38: 409-421, 1978; Ryberg, B., J. Neurol. Sci. 54: 239-261, 1982. ). There is also clear evidence that complement plays a prominent role in the pathophysiology of demyelinating CNS or PNS disorders including MS, Guillain-Barre syndrome, and Miller-Fisher syndrome (Gasque, P., et al., Immunopharmacology49: 171-186, 2000 Barnum, SR in Bondy S. et al. (Eds.) Inflammatoryevents in neurodegeration, Prominent Press 139-156, 2001). Complementary contributes to tissue destruction, inflammation, clearance of demyelin fragments and even remyelination of axons. Despite clear evidence of complement development, the identification of target-complement therapeutic products is only now being evaluated in experimental animal encephalomyelitheality (EAE), an animal model of C3 or factor B deficient EAE mice showing attenuated demyelination when compared to EAE control mice (Barnum, Immunologic Research 26: 7-13, 2002). Studies in EAE mice using a soluble form of a sCrry and C3 - / - and factor B - / - coined inhibitor complement showed that the complement contributes to the development and progression of the fingerprint model at various levels. In addition, the marked reduction in the severity of EAE in factor B - / - mice provides further evidence for the role of the alternative complement pathway in EAE (Nataf et al., S. Immunology 165: 5867-5873, 2000).

MG is a neuromuscular junction disease with an acetylcholine receptors peril and end plate destruction. sCRl is very effective in an animal model of MG, further indicating its role in disease (Piddelesden et al., J. Neuroimmunol. 1997).

The histological marks of AD, a neurodegenerative disease, are senile plaques and neurofibrillary tangles (McGeer et al., Res. Immunol.143: 621-630, 1992). These pathological markers also strongly dye the complement system components. Evidence indicates a local neuroinflammatory state that results in neuronal death and cognitive impairment. Senile plaques contain abnormal amyloid-0 peptide (AG, a peptide derived from the amyloid precursor protein. AD shown to bind Cle may trigger complement activation (Rogers et al., Res. Immunol. 143: 624-630, 1992). Also, a prominent feature of AD is the association of activated proteins from the classic complement pathway of Clq to C5b-9, which has been found to be highly localized in neuritic plaques (Shen, Y., et al., Brain Research 769: 391-395, 1997; Shen , Y., etL, Neurosci Letters 305 (3): 165-168, 2001) Thus, AD not only initiates the classical pathway, but a resultant continuous inflammatory state can contribute to neuronal cell death. that complement activation in AD progressed to the terminal C5b-9 phase indicating that the regulatory mechanisms of the complement system were unable to arrest the complement activation process.

Several complement pathway inhibitors have been proposed as potential therapeutic methods for AD, including proteoglycans such as ClQ binding inhibitors, Nafamstat as a C3 convertase inhibitor and C5 activation blockers or C5a receptor inhibitors (Shen, Y., et al., Progress in Neurobiology 70 : 463-472, 2003). The role of MASP-2 as an initiating step in the innate complement pathway, as well as for the activation of the alternative pathway, provides a potentially new therapeutic method and is supported by the abundance of data suggesting complement pathway involvement in AD.

In regions damaged in the brains of PD patients, as with other degenerative CNS diseases, there is evidence of inflammation characterized by glial reaction (especially microglia), as well as increased expression of HLA-DR antigens, cytokines, and decomposition components. These observations suggest that immune system mechanisms are involved in the pathogenesis of neuronal injury in PD. Cellular mechanisms of primary PD injury have not been clarified, but it is, however, likely that mitochondrial mutations, oxidative stress and apoptosis play a role. In addition, inflammation initiated by striatal neuronal skin damage and the substantia nigra in PD may aggravate the disease course. These observations suggest that treatment with complement-inhibiting drugs may act to slow the progression of PD (Czlonkowska, A., et al., Med. Sci. Monit. 8: 165-177, 2002).

One aspect of the invention is thus directed to the treatment of disorders or injuries of the peripheral nervous system (PNS) and / or central nervous system (CNS) by treating a patient suffering from such a disorder or injury with a composition comprising a therapeutically effective amount of an agent. inhibitor in a pharmaceutical carrier. It is believed that CNS and PNS disorders and lesions that can be treated according to the present invention include but are not limited to multiple sclerosis (MS), myasthenia gravis (MG), Huntington (HD), Amyotrophic lateral sclerosis (ALS), Guillain Barre syndrome, reperfusion following stroke, degenerative discs, brain trauma, Parkinson's disease (PD), Alzheimer's disease (AD), Miller-Fisher syndrome, trauma and / or cerebral hemorrhage, demyelination, and possibly meningitis.

For the treatment of CNS conditions and brain trauma, the MASP-2 inhibitor agent may be administered to the patient by intrathecal, intracranial, intraventricular, intraarterial, intravenous, intramuscular, subeutaneous or other potential parenteral administration orally to non-inhibitors. peptidergic. PNS and brain trauma conditions may be treated by a systemic route of administration or alternatively by local administration to the dysfunction or trauma site. Administration of the MASP-2 inhibitor compositions of the present invention may be repeated periodically as determined by a physician until effective relief or control of symptoms is obtained. BLOOD DISORDERS

Septicemia is caused by an overwhelming patient reaction to invading microorganisms. A major function of the breakdown system is to orchestrate the inflammatory response to invasive bacteria and other pathogens. Compatible with this physiological role, activation of complement has been shown in numerous studies to have a major role in the pathogenesis of septicemia (Bone, R. C., Annals. Internai. Med. 115: 457-469, 1991). The definition of clinical manifestations of septicemia is always evolving. Septicemia is usually defined as the systemic host response to an infection. However, on many occasions no clinical evidence for infection (eg, positive bacterial blood cultures) is found in patients with septic symptoms. This discrepancy was first accounted for at a 1992 Consensus Conference when the term "systemic inflammatory response syndrome" (SIRS) was established and for which no definable presence of bacterial infection was required (Bone, RC, et al., Crit. Care Med. 20: 724-726, 1992). There is now general agreement that septicemia and SIRS are accompanied by the ability to regulate the inflammatory response. For the purposes of this slight review, we will consider the clinical definition of septicemia including also severe septicemia, septic shock and SIRS.

The predominant source of infection in septic patients before the 1980s was Gram-negative bacteria. Lipopolysaccharide (LPS), the major component of the gram-negative bacterial cell wall, has been known to stimulate the release of inflammatory mediators from various cell types and to induce acute infectious symptoms when injected emanimal (Haeney, MR, et al., Antimicrobial Chemotherapy 41). (Suppl. A): 4th, 1998). Interestingly, the spectrum of responsible microorganisms appears to have shifted from predominantly Gram-negative bacteria in the 1970s and 1980s to predominantly Gram-positive bacteria at present, for reasons that are currently uncertain (Martin, GS, et al., N. Eng. J. Med. 348: 1546-54, 2003).

Many studies have shown the importance of activation-decomposition in mediating inflammation and contributing to stroke features, particularly septic and hemorrhagic shock. Both Gram-negative and Gram-positive organisms usually precipitate shock shock. LPS is a potent complement activator, predominantly by the alternative pathway, although antibody-mediated classic pathway activation also occurs (Fearon, D. T., et al., N. Engl. J.

Med. 292: 937-400, 1975). The major components of the Gram-positive cell wall are peptidoglycan and lipoteichoic acid and both components are potent activators of the alternative complement pathway, although in the presence of specific antibodies they may also activate the classical complement pathway (Joiner, Κ. A., et al., Ann, Rev. Immunol. 2: 461-2, 1984).

The complement system was initially implicated in the pathogenesis of septicemia when it was noted by the researchers that asanafilatoxins C3a and C5a mediate a variety of inflammatory reactions that could also occur during septicemia. These anaphylatoxins evoke avasodilation and an increase in vascular micropermeability, events that play a central role in septic shock (Schumacher, W.A., et al., Agents Actions 34: 345-349, 1991). In addition, anaphylatoxins induce spasm, histamine release from mastoids and platelet aggregation. In addition, they exert numerous effects on granulocytes, such as chemotaxis, aggregation, adhesion, lysosomal enzyme release, toxic superoxide anion generation, and leukotriene formation (Shin, HS, etal., Science 162: 361-363, 1968 ; Vogt, W., Complement 3: 177-86, 1986). These biological effects are considered to play a role in the development of complications of septicemia such as shock or acute respiratory distress syndrome (ARDS) (Hammerschmidt, DE, etal., Lancet 1; : 947-949, 1980; Slotman, GT, et al., Surgery 99: 744-50,1986). In addition, elevated levels of anaphylatoxin C3a are associated with a fatal outcome in septicemia (Hack, C.E., et al., Am. J. Med. 86: 20-26,1989). In some animal models of shock, certain complement-deficient strains (e.g., those deficient in C5) are more resistant to the effects of LPS infusions (Hseuh, W., et al., Immunol. 70: 309-14,1990).

Blocking C5a Generation with Antibodies During Early Septicemia in Rodents has been shown to greatly improve survival (Czermak, BJ, et al., Nat. Med. 5: 788-792, 1999). Similar findings were made when the C5a receptor (C5aR) was blocked with antibodies or with a small molecular inhibitor (Huber-Lang, MS, et al., FASEB J. 16: 1567-74, 2002; Riedemann, NC, et al., J.Clin. Invest. 110: 101-8, 2002). Older experimental studies in monkeys have suggested that C5a antibody block attenuated E. coli-induced shock and adult respiratory distress syndrome (Hangen, DH, et al., J. Surg. Res. 46: 195-9 , 1989; Stevens, JH, et al., J.Clin. Invest. 77: 1812-16, 1986). In humans with septicemia, C5a was elevated and associated with significantly reduced survival rates along with multiple organ failure when compared with those in less severely septic patients and survivors (Nakae, H., et al., Res. Commun. Chem. Pathol Pharmacol 84: 189-95,1994; Nakae, et al., Surg. Today 26: 225-29, 1996; Bengtson, A., et al., Arch.Surg. 123: 645-649, 1988) . The mechanisms by which C5a exerts its deleterious effects during septicemia are yet to be investigated in greater detail, but recent data suggest that C5a generation during asepticemia significantly compromises the innate immune functions of blood deneutrophils (Huber-Lang, MS, et al., J Immunol 169: 3223-31,2002), their ability to express a respiratory outbreak and their ability to generate cytokines (Riedemann, NC, et al., Immunity 19: 193-202, 2003). Furthermore, generation of C5a during septicemia appears to be procoagulant terefects (Laudes, I.J., et al., Am. J. Pathol. 160: 1867-75,2002). CI INH complement modulating protein has also been shown to be effective in animal models of septicemia and ARDS (Dickneite, G., BehringIns. Mitt. 93: 299-305, 1993).

The lectin pathway may also play a role in the sepatogenesis of septicemia. MBL has been shown to bind to a range of clinically important micro-organisms including both Gram-negative and Gram-positive bacteria and to activate the lectin pathway (Neth, O., etal., Infect. Immun. 68: 688, 2000). Lipoteichoic acid (LTA) is increasingly considered to be the gram-positive counterpart of LPS. This is a potent immunostimulant that induces cytokine release from mononuclear phagocytes and whole blood (Morath, S. et al., J. Exp. Med. 195: 1635,2002; Morath, S. et al., Infect. Immun. 70: 938, 2002). L-phycoline has recently been shown to specifically bind isolated LTA to numerous Gram-positive bacterial species including Staphylococcusaureus and activate the lectin pathway (Lynch, NJ, et al., J. Immunol. 172: 1198-02, 2004) . MBL has also been shown to bind to the LTA of

Enterocoeeus spp where the polyglycerophosphate chain is substituted with glycosyl groups), but not to the LTA of nine other species including S.aureus (Polotsky, V. Y., et al., Infect. Immun. 64: 380, 1996).

One aspect of the invention thus provides a method for treating septicemia or a condition resulting from septicemia by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibiting agent in a pharmaceutical carrier to a patient suffering from septicemia or a condition suffering from septicemia. results from septicemia including without limitation severe septicemia, septic shock, acute respiratory distress syndrome resulting from septicemia, and systemic inflammatory response syndrome. Related methods are provided for the treatment of other blood disorders, including haemorrhagic shock, haemolytic anemia, autoimmune thrombotic thrombocytopenic purpura (TTP), haemolytic uremic syndrome (HUS), or other destructive damedula / blood conditions, by administering a composition comprising a therapeutically effective amount. of a MASP-2 inhibiting agent in a pharmaceutical vehicle to a patient suffering from such a condition. The MASP-2 inhibitor is administered to the patient systemically, such as by intraarterial, intravenous, intramuscular, inhalational (particularly in the case of ARDS), subcutaneous or other parenteral administration or potentially by oral administration for non-peptide agents. The MASP-2 composition inhibitory agent may be combined with one or more additional therapeutic agents to combat the effects of septicemia and / or shock. For advanced septicemia or shock or a distress condition resulting therefrom, the DEMASP-2 inhibitor composition may be suitably administered in a rapid-dose dosage form, such as by intravenous or intraarterial release of a bolode into a solution containing the composition. of MASP-2 inhibiting agent. Repeated administration may be performed as determined by a physician until the condition has been resolved.

UROGENITAL CONDITIONS

The complement system has been implicated in several distinct urogenital disorders including painful bladder disease, sensitive bladder disease, chronic nonbacterial cystitis, and interstitial cystitis (Holm-Bentzen, M., et al., J. Urol. 138: 503-507, 1987), infertility (Cruz, et al., Biol. Reprod. 54: 1217-1228, 1996), pregnancy (Xu, C., et al., Science 287: 498-507, 2000), fetal-maternal tolerance (Xu, C., et al., Science 287: 498-507,2000) and preeclampsia (Haeger, M., Int. J. Gynecol. Obstet. 43: 113-127,1993).

Painful bladder disease, sensory bladder disease, chronic nonbacterial cystitis, and interstitial cystitis are conditions defined by a variety of unknown etiology and pathogenesis, and therefore are without any rational therapy. Pathogenetic theories with respect to bladder epithelial and / or mucosal surface coat defects and theories with respect to immunological disorders predominate (Holm-Bentzen, M., et al., J. Urol. 138: 503-507, 1987). Patients with interstitial cystitis have been tested for immunoglobulins (IgA, G, M), complement components (Clq, C3, C4) and Cl-esterase inhibitor. There was a highly significant depletion of serum levels of component C4 (p <0.001) and the immunoglobulin G was significantly higher (p <0.001). This study suggests activation of the classical pathway complement system and supports the possibility that a chronic local immune process is involved in the disease pathogenesis (Mattila, J., et al., Eur. Urol. 9: 350-352, 1983). In addition, following binding of autoantibodies to bladder mucosa antigens, complement activation would be involved in the production of tissue damage and chronic self-perpetuating inflammation of this disease (Helin, H., et al., Clin. Immunol. Immunopathol 43: 88-96, 1987).

In addition to the complement role in urogenital inflammatory diseases, reproductive functions may be impacted by local regulation of the complement pathway. Naturally occurring complement inhibitors have evolved to provide host cells with the protection they need to control the body's complement system. Crry, a naturally occurring rodent complement inhibitor that is structurally similar to human complement inhibitors, MCP and ADF, has been investigated to delineate regulatory regulatory control of fetal development. Interestingly, attempts to generate Crry - / - mice were unsuccessful. Instead, it was discovered that homozygous Crry - / - mice died in the womb. Crry - / - Embryos survived until about 10 days after intercourse, and survival quickly declined with death resulting from arrest of development. There was also a marked invasion of inflammatory cells in the placental tissue of Crry - / - embryos. In contrast, Crry + / + embryos appeared to have C3 deposited in the placenta. This suggests that complement activation occurred at the placenta level and in the absence of complement regulation, the embryos died. Confirmatory studies investigated the introduction of the Crry mutation in a C3 deficient background.

This rescue strategy was successful. Together, these data illustrate that the fetal complement interface should be regulated. Subtle changes in complement complement within the placenta could contribute to placental dysfunction and abortion (Xu, C., et al., Science 287: 498-507, 2000).

Preeclampsia is a pregnancy-induced hypertensive disorder in which activation of the complement system has been implicated but remains controversial (Haeger, M., Int. J. Gynecol. Obstet. 43: 113-127,1993). Complement activation in the systemic circulation is closely related to the disease established in preeclampsia, but no elevation was observed prior to the presence of clinical symptoms, and therefore complement components cannot be used as predictors of preeclampsia (Haeger, et al. , Obstet, Gynecol, 78: 46, 1991). However, increased complement activation in the local environment of the placental bed could overcome local control mechanisms resulting in elevated levels of anaphylatoxins and C5b-9 (Haeger, et al., Obstet. Gynecol. 73: 551,1989).

A proposed mechanism of antisperm-related antibody (ASA) related infertility is through the role of activation of the genital tract complement. Generation of C3b and iC3b opsonin, which may potentiate sperm alleviation by phagocytic cells through their complement receptors as well as the formation of the C5b-9terminal complex on the sperm surface, thereby reducing doesperm motility, are potential causes associated with reduced fertility. Elevated C5b-9 levels have also been demonstrated in follicularovarian fluid of infertile women (D'Cruz, O. J., et al., J. Immunol. 144: 3841-3848, 1990). Other studies have shown deterioration in doesperm migration and reduced sperm / egg interactions, which may be associated with complement (D'Cruz, OJ, et al., J. Immunol. 146: 611-620, 1991; Alexander, NJ, Fertile. Steril 41: 433-439, 1984). Finally, studies with CRS demonstrated a protective effect against ASA-mediated injury and complement to human sperm (D'Cruz, O. J., et al., Biol. Reprod. 54: 1217-1228, 1996). These data provide several lines of evidence for the use of complement inhibitors in the treatment of urogenital disorders and disorders.

One aspect of the invention thus provides a method for inhibiting MASP-2-dependent complement activation in a patient suffering from a urogenital disorder by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibiting agent in a pharmaceutical carrier to a patient suffering from a taldisturbia. Urogenital disorders to be treated with the methods and compositions of the present invention are believed to include, by way of non-limiting example, painful bladder disease, sensitive bladder disease, chronic non-bacterial cystitis and interstitial cystitis, male and female infertility, placental dysfunction and abortion and preeclampsia. The MASP-2 inhibitory agent may be administered to the patients systematically, such as by intraarterial, intravenous, intramuscular, inhalation, subcutaneous or other parenteral administration or potentially by oral administration to non-peptidergic agents. Alternatively, the MASP-2 inhibitor composition 2 may be released locally to the urogenital tract, such as by intravesical irrigation or instillation with a liquid solution or gel composition. Repeated administration may be performed as directed by a physician to control or resolve the condition.

DIABETIC AND DIABETIC CONDITIONS

Diabetic retinal microangiopathy is characterized by increased permeability, leukostasis, microthrombosis and apoptosis of capillary cells, all of which would be caused or promoted by complement activation. Diabetes-depleting glomerular structures and endoneurial micro vessels show signs of complement activation. Decreased availability or efficacy of nadiabete complement inhibitors has been suggested by the findings that high in vitro glucose selectively decreases CD55e expression on the endothelial cell surface. by non-enzymatic glycation that prevents its complement inhibitory function.

Studies by Zhang et al (Diabetes 51: 3499-3504, 2002) investigated complement activation as a trait of non-human proliferative diabetic retinopathy and its association with changes in inhibitor molecules. C5b-9 deposition, the terminal product of complement activation, has been found to occur in the retinal vessel wall of human eye donor donors with type-2 diabetes, but not in the vessels of age-matched non-diabetic donors. Clq and C4, the complement components unique to the classical pathway, were not detected in diabetic retinas, indicating that C5b-9 was generated via the alternative pathway. Diabetic donors showed a prominent reduction in retinal levels of CD55 and CD59, the two plasma membrane-bound complement inhibitors by GPI anchors. Complement activation in retinal vessels and selective reduction in similar retinal CD55 and CD59 levels were observed in rats with a 10-week duration of streptozotocin-induced diabetes. Thus, diabetes appears to cause poor regulation of complement inhibitors and complement activation that precedes most other manifestations of diabetic retinal microangiopathy.

Gerl et al. (Investigative Ophthalmology and Visual Science43: 1104-08, 2000) determined the presence of activated decomposition components in the eyes affected by diabetic retinopathy. Immunohistochemical studies have found extensive deposits of C5b-9 decomplexing complexes that were detected in the coriocapillary immediately underlying Bruch's membrane and densely surrounding the capillaries in all 50 specimens of diabetic retinopathy. Dyeing for C3d positively correlated with C5b-9 dyeing, indicative of the fact that complement activation occurred in situ. In addition, positive dyeing has been discovered for vitronectin, which forms stable complexes with extracellular C5b-9. In contrast, there was no positive staining for C-reactive protein (CRP), mannan-binding lectin (MBL), Clq or C4, indicating that complement activation did not occur via a C4-dependent pathway. Thus, the presence of C3d, C5b-9 and vitronectin indicates that complement activation occurs until completion, possibly via the alternative pathway in the coriocapillaries of eyes affected by diabetic retinopathy. Complement activation may be a causative factor in sequelae that may contribute to ocular tissue disease and visual deterioration. Therefore, the use of a complement inhibitor may be an effective therapy to reduce or block the microvessel damage that occurs with diabetes.

Insulin-dependent diabetes mellitus (IDDM, also referred to as Type-1 diabetes) is an autoimmune disease associated with the presence of different types of autoantibodies (Nicoloff et al., Clin. Dev.Immunol. 11: 61-66, 2004). The presence of these antibodies and their corresponding antigens in the circulation leads to the formation of immunoscirculatory complexes (CIC), which are known to persist in the blood for long periods of time. CIC deposition in small blood vessels has the potential to lead to microangiopathy with debilitating clinical consequences. A correlation exists between CIC and the development of microvascular complications in diabetic children. These findings suggest that elevated levels of CIC IgG are associated with the development of early diabetic nephropathy and that a complement pathway inhibitor may be effective in blocking diabetic nephropathy (Kotnik, et al., Croat.Med. J. 44: 707-11 , 2003). In addition, downstream complement protein formation and alternative pathway involvement is likely to be a contributing factor in global islet cell function in IDDM and use of a complement inhibitor to reduce potential injury or limit cell death is expected ( Caraher, et al., J. Endocrinol, 162: 143-53, 1999).

Circulating MBL concentrations are significantly elevated in type 1 diabetes patients compared with healthy ones, and these MBL concentrations positively correlate with urinary albumin excretion (Hansen et al., J. Clin. Endocrinol. Metab.88: 4857-61, 2003). A recent clinical study found that the frequencies of high and low expression MBL genotypes were similar between type 1 diabetes patients and healthy controls (Hansen et al., Diabetes53: 1570-76, 2004). However, the risk of having nephropathy among patients with diabetes was significantly increased if they had a high MBL genotype. This indicates that high MBL levels and complemental activation of the lectin pathway may contribute to the development of diabetic nephropathy. This conclusion is supported by a recent prospective study where the association between MBL levels and the development of albuminuria in a group of patients with newly diagnosed type 1 diabetes mellitus (Hovind et al., Diabetes 54: 1523-27, 2005). They found high levels of premature MBL in the course of type 1 diabetes were significantly associated with the subsequent development of persistent albuminuria. These results suggest that MBL and the dalectin pathway may be involved in the specific pathogenesis of diabetic vascular complications rather than merely causing an acceleration of existing changes. In a recent clinical study (Hansen et al., Arch.Intern. Med. 166: 2007-13, 2006), MBL levels were measured at baseline in a well-characterized group of patients with type 2 diabetes who received more than 15 years of follow up. They found that even after adjusting for known confounders, the risk of dying was significantly higher among patients with plasma MBLalt levels (> 1000 μg / L) than among patients with low MBL levels (<1000μg / L).

In another aspect of the invention, methods are provided for inhibiting MASP-2-dependent complement activation in a patient suffering from nonobese diabetes mellitus (IDDM) or complications of adult-onset diabetes mellitus, neuropathy or retinopathy (Type ID). -2) by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical vehicle. The MASP-2 inhibitory agent may be administered to the patient systemically, such as by intraarterial, intravenous, intramuscular, subcutaneous or other parenteral administration or potentially by oral administration to non-peptideric agents.

Alternatively, administration may be by local release to the site of angiopathic, neuropathic or retinopathic symptoms. The MAPS-2 inhibitory agent may be administered periodically over an extended period of time for the treatment or control of a chronic condition or for a single administration or series of administrations for the treatment of an acute condition.

PERIQUIOLOGICAL ADMINISTRATION AND TREATMENT DEMALIGNI PAPES

Activation of the complement system may also be implicated in the pathogenesis of malignancies. Recently, the complement complement C5b-9, IgG, C3, C4, S / vitronectin protein, fibronectin, and macrophage neoantigens were located in 17 breast cancer samples and 6 benign breast tumor samples using polyclonal or monoclonal antibodies and the technique. streptavidin-biotin-peroxidase.All samples of carcinoma tissue in each of the TNM stages showed C5b-9 deposits on tumor cell membranes, fine granules in cell debris, and diffuse deposits in necrotic areas (Niculescu, F., et al., Am. J. Pathol. 140: 1039-1043, 1992).

In addition, complement activation may be a consequence of chemotherapy or radiation therapy and thus inhibition of complement activation would be useful as an adjunct in the treatment of malignancies to reduce iatrogenic inflammation. When chemotherapy and radiation therapy preceded surgery, C5b-9 deposits were more intense and enlarged. C5b-9 deposits were absent in all samples with benign lesions. Protein S / vitronectin was present as fibrillar deposits in the connective tissue matrix and as diffuse deposits around tumor cells, less intense and enlarged than afibronectin. IgG, C3 and C4 deposits were present only in carcinoma samples. The presence of C5b-9 deposits is indicative of complement activation and its subsequent pathogenic effects on breast cancer (Niculescu, F., et al., Am. J. Pathol. 140: 1039-1043, 1992).

Harmonized pulsed pigment (577 nm) laser therapy (PTDL) induces hemoglobin coagulation and tissue necrosis, which is mainly limited to blood vessels. In a study of normal PTDL-irradiated skin, the main findings were as follows: 1) C3, C8, C9 and MAC fragments were deposited on the vessel walls; 2) these deposits were not due to protein denaturation as the same became evident only 7 min after irradiation, contrary to immediate transferrin deposition at the erythrocyte clot sites; 3) C3 deposits were shown to amplify complement activation by the alternative pathway, a reaction that was specific since tissue necrosis itself did not lead to such amplification; and 4) these reactions preceded the local accumulation of polymorphonuclear leukocytes. Tissue necrosis was more pronounced in hemangiomas. Larger angiomatous vessels in the center of danecrosis did not significantly fix complement. In contrast, complement compliance in peripheral vessels was similar to that observed in normal skin with one exception: C8, C9, and MAC were detected in some blood vessels immediately after laser treatment, a finding compatible with MAC assembly that occurs directly in formation of a C5 convertase. These results indicate that complement is activated in PTDL-induced vascular necrosis and is responsible for resulting inflammatory response.

Photodynamic therapy (PDT) of tumors evokes a strong host immune response and one of its manifestations is a pronounced neutrophilia. In addition to complement fragments (direct mediators) released as a consequence of PDT-induced complement activation, there are at least a dozen secondary mediators that all arise as a result of complement activity. The latter include cytokines IL-I beta, TNF-alpha, IL-6, IL-10, G-CSF and KC, thromboxane, prostaglandins, leukotrienes, histamine and coagulation factors (Cecic, I., etal., CancerLett. 183: 43-51, 2002).

Finally, the use of MASP-2-dependent complement activation inhibitors may be envisaged in conjunction with the standard regimeter therapeutic for cancer treatment. For example, treatment with rituximab, a chimeric anti-CD20 monoclonal antibody, may be associated with moderate to severe first dose side effects, notably in patients with high numbers of circulating tumor cells. Recent studies during the first rituximab infusion measured the activation of complement products (C3b / c and C4b / c) and cytokines (alpha tumor necrosis factor (TNF-alpha), interleukin 6 (IL-6) and IL-8) in five patients with low-grade non-Hodgkin's lymphoma (NHL). Rituximab infusion induced rapid decompression activation, preceding the release of TNF-alpha, IL-6 and IL-8. Although the study group was small, the level of complement activation appeared to correlate with both the number of circulating B cells before infusion (r = 0.85; P = 0.07) and the severity of side effects. The results indicated that complement plays a central role in the pathogenesis of side effects of rituximab treatment. As complement activation cannot be prevented by corticosteroids, it may be relevant to study the possible role of complement inhibitors during the first administration of rituximab (van der Kolk, LE, et al., Br. J. Haematol. 115: 807-811, 2001 ).

In another aspect of the invention, methods are provided for inhibiting MASP-2 dependent complement activation in a patient who is treated with chemotherapeutic products and / or radiation therapy, including without limitation for the treatment of cancerous conditions.

This method includes administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical vehicle to a peri-chemotherapeutic patient, i.e. anthesis / or during and / or after administration of chemotherapeutic product (s) and / or radiation therapy. For example, administration of a MASP-2 inhibitory composition of the present invention may be initiated prior to or concurrently with administration of chemotherapy or radiation therapy and continued throughout the course of therapy to reduce the harmful effects of chemotherapy and / or radiation therapy on unbleached healthy tissues. In addition, the MASP-2 inhibitor composition may be administered following chemotherapy and / or radiation therapy. It is understood that chemotherapy and radiation therapy regimens often entail repeated treatments and, therefore, it is possible that administration of a MASP-2 inhibitor composition will also be repetitive and coincidentally with chemotherapeutic and radiation treatments. MASP-2 inhibiting agents are also believed to be used as chemotherapeutic agents, alone or in combination with other chemotherapeutic agents and / or radiation therapy, to treat patients suffering from malignancies. Administration may be suitably by oral (non-peptidergic), intravenous, intramuscular or other parenteral route. ENDOCRINE DISORDERS

The complement system has also recently been associated with a few endocrine conditions or disorders including Hashimoto's thyroiditis (Blanchin, S., et al., Exp. Eye Res. 73 (6): 887-96,2001), stress, anxiety, and other disorders. Potential hormonal hormones involve the regulated release of prolactin, growth factor, or growth factor equivalent to pituitary insulin and adrenocorticotropin (Francis, K., et al., FASEB J. 17: 2266-2268, 2003; Hansen, TK, Endocrinology 144 ( 12): 5422-9, 2003).

Two-way communication exists between the endocrine and immune systems using molecules such as hormones and cytokines. Recently, a new pathway has been elucidated by which C3a, a decomposition-derived cytokine, stimulates the release of the anterior pituitary hormone and activates the hypothalamic-pituitary-adrenal axis, a central reflex for stress response and inflammation control. C3a receptors are expressed on cells that secrete pituitary hormone and cells that do not secrete hormone (follicular star). C3a and C3adesArg (a noninflammatory metabolite) stimulate pituitary cell cultures to release deprolactin, growth hormone and adrenocorticotropin. Serum levels of these hormones, together with adrenal corticosterone, increase dose-dependent with in vivo administration of C3a and C3adesArgrecombinant. The implication is that the complement pathway modulates tissue-specific and systemic inflammatory responses through communication with the endocrine pituitary gland (Francis, K., et al., FASEBJ. 17: 2266-2268, 2003).

A growing number of animal and human studies indicate that growth hormone (GH) and insulin-equivalent growth factor (IGF-I) modulate immune function. GH therapy increased mortality in critically ill patients. Excessive mortality was almost entirely due to septic shock or multiple organ failure, which could suggest that GH-induced modulation of immune and complement function was involved. MBL is a plasma protein that plays an important role in innate immunity by activating the complement cascade and inflammation following binding to carbohydrate structures. Evidence bears a significant influence of growth hormone on MBL levels and therefore potentially on lectin-dependent complement activation (Hansen, T. K., Endocrinology 144 (12): 5422-9, 2003).

Thyroperoxidase (TPO) is one of the major autoantigens involved in autoimmune thyroid disease. TPO consists of a large N-terminal myeloperoxidase equivalent module followed by a complementary control protein equivalent (CCP) equivalent module and an epidermal growth factor equivalent module. The CCP module is a constituent of the molecules involved in activation of the C4 breaker component and studies have been conducted to investigate whether C4 binds to TPO and activates the complement pathway under autoimmune conditions.

TPO via its directly active CCP module complements no mediation by Ig. In addition, in patients with Hasimoto thyroiditis, thyrocytes overexpress C4 and all components of the downstream complement pathway. These results indicate that TPO, along with other mechanisms related to activation of the pathway, may contribute to the massive cellular destruction observed in Hashimoto's thyroiditis (Blanchin, S., et al., 2001).

One aspect of the invention thus provides a method for inhibiting MASP-2-dependent complement activation to treat an endocrine disorder by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibiting agent in a pharmaceutical vehicle to a patient suffering from an endocrine disorder. Patient conditions for treatment in accordance with the present invention include, by way of non-limiting example, Hashimoto's thyroiditis, stress, anxiety, and other potential hormonal disorders involving regulated release of prolactin, growth factor, or insulin-equivalent growth factor. pituitary adrenocorticotropin. The MAS-2 inhibitory agent may be administered to the patient systemically, such as by intraarterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral administration or potentially by oral administration to non-peptidergic agents. The MASP-2 inhibitor composition may be combined with one or more additional therapeutic agents. Administration may be repeated as determined by a physician until the condition has been resolved.

Ophthalmological conditions Age-related macular degeneration (AMD) is a blind disease that afflicts millions of adults, despite the biochemical, cellular, and / or molecular sequelae leading to the development of AMD are poorly understood. AMD results in the progressive destruction of the macula that has been correlated with the formation of extracellular deposits called drusen located in and around the macula, behind the retina and between the retinal pigment epithelium (RPE) and the choroid. Recent studies have revealed that proteins associated with inflammation and immune-mediated processes are prevalent among the constituents associated with drusen. Transcripts encoding several of these molecules were detected in retinal, RPE and choroidal cells. These data also demonstrate that dendritic cells, which are potent antigen-presenting cells, are closely associated with blunt development and that complement activation is a key pathway that is active throughout the drusen along the RPE-choroidal interface (Hageman, GS, et al., Prog. Retin. Eye Res., 20: 705-732 (2001).

Several independent studies have shown a strong association between AMD and a gene polymorphism in the factor H-decomposition (CFH) gene in which the probability of AMD is increased by a factor of 7.4 in homozygous individuals for risk allele (Klein, RJet al., Science, 308: 362-364, 2005; Haines et al., Science 308: 362-364.2005; Edwards et al., Science 308: 263-264, 2005). The CFH gene has been mapped to chromosome lq31, a region that has been implicated in AMD by six independent binding scans (see, for example, D. W. Schultz et al., Hum. Mol. Genet. 12: 3315, 2003). CFH is known to be a key regulator of the complement system. CFH in cells and nacirculation has been shown to regulate complement activity by inhibiting C3 activation for C3a and C3b and by existing C3b inactivation. C5b-9 deposition was observed in the Brusch membrane, intercapillary pillars, and within dadrusa in AMD patients (Klein et al.). Immunofluorescence experiments suggest that in AMD, CFH polymorphism may give rise to complement deposition in choroidal capillaries and choroidal vessels (Klein et al.).

The membrane-associated complement inhibitor, complement receptor 1, is also located on the drusen, but it is not detected in immunohistochemically RPE cells. In contrast, a second membrane-associated complement inhibitor, demembrane cofactor protein, is present in drus-associated RPE cells as well as small, spherical substructural elements within the drusen. These previously unidentified elements also show strong immunoreactivity to proteolytic fragments of complement component C3 which are characteristically deposited at complement activation sites. These structures are proposed to represent residual degenerating RPE cell fragments that are targets of complement attack (Johnson, L. V., et al., Exp. Eye Res. 73: 887-896, 2001).

The identification and localization of these multiple-complement regulators as well as the activation of complement products (C3a, C5a, C3b, C5b-9) have led researchers to conclude that chronic complement activation plays an important role in the process of druze biogenesis and etiology of AMD (Hageman et al., Progress Retinal Eye Res. 20: 705-32, 2001). The identification of C3 and C5 activation products in the drusen provides no insight as to whether complement is activated via the classical pathway, lectin pathway or alternative amplification loop as understood in accordance with the present invention, since both C3 as well as C5 are common to all three.However, two studies have observed for immuno-labeling of drusa using Clq-specific antibodies, the essential recognition component for classic pathway activation (Mullins et al., FASEB J. 14: 835 -846, 2000; Johnson et al., Exp. Eye Res. 70: 441-449, 2000). Both studies concluded that Clq immuno-labeling in drusen was not generally observed. These negative results with Clq suggest that activation of the druse's complement does not occur through the classical pathway. In addition, drusen immuno-labeling of the immune complex constituents (IgG light chains, IgM) is reported in the study by Mullins et al., 2000 to be weak to variable, further indicating that the classical pathway plays a minor role in complement activation that occurs in this disease process.

Two recent published studies have evaluated the role of complement in the development of laser-induced choroidal neovascularization (CNV) in mice, a human CNV model. Using immunohistological methods, Bora and colleagues (2005) found significant deposition of activation of complement products C3b and C5b-9 (MAC) in the neovascular complex following laser treatment (Bora et al., J. Immunol. 174: 491-7, 2005). Importantly, CNV does not develop genetically C3-deficient mice (C3 - / - mice), the essential component required in all complement-activation pathways. RNA message levels for VEGF, TGF-P2 and β-FGF, three angiogenic factors implicated in CNV, were elevated in mouse eye tissue after laser-induced CNV. Significantly, complement depletion resulted in a marked reduction in RNA levels of these angiogenic factors.

Using ELISA methods, Nozaki and colleagues demonstrated that potent C3a and C5a anaphylatoxins are generated early in the course of laser-induced CNV (Nozaki et al., Proc. Natl. Acad. Sci. U.S.A. 103: 2328-33, 2006). In addition, these two C3 and C5 bioactive fragments induced VEGF expression following intravitreal injection into wild type mice. Compatible with these results Nozaki echolegas also showed that gene ablation of C3a and C5a receptors reduces VEGF expression and CNV formation after porlaser injury and that C3a or C5a antibody-mediated neutralization or pharmacological obliteration of their receptors also reduces the CNV. Previous studies have established that leukocyte and macrophage recruitment in particular plays a central role in laser-induced CNV (Sakurai et al., Invest. Opthomol. Vis. Sci. 44: 3578-85, 2003; Espinosa-Heidmann, et al. Invest, Opthomol, Vis. Sci. 44: 3586-92, 2003). In their 2006 paper, Nozaki and colleagues report that delukocyte recruitment is markedly reduced in C3aR (- / -) and C5aR (- / -) mice following laser injury.

One aspect of the invention thus provides a method for inhibiting MASP-2-dependent complement activation for treating age-related muscle degeneration or other complement-mediated ophthalmic condition by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibiting agent. a pharmaceutical carrier to a patient suffering from such a condition or other complement-mediated ophthalmic condition. The MASP-2 inhibitor composition may be administered locally to the eyes, such as by irrigation or application of the composition in the form of a gel, ointment or fillets. Alternatively, the MASP-2 inhibitory agent may be administered to the patient systemically, such as by intraarterial, intravenous, intramuscular, inhalation, nasal, subeutaneous or other parenteral administration or potentially by oral administration to non-peptideric agents. The MASP-2 inhibitor agent composition may be combined with one or more additional therapeutic agents, such as are disclosed in U.S. Patent Application Publication No. 2004-0072809-A1. Administration may be repeated as determined by a physician until the condition has been resolved or controlled. IV. MASP-2 INHIBITOR AGENTS

In one aspect, the present invention provides methods of inhibiting adverse effects of MASP-2 dependent complement activation. MASP-2 inhibitor agents are administered in an amount effective to inhibit MASP-2-dependent complement activation in a living patient. In practicing this aspect of the invention, representative DEMAS-2 inhibiting agents include: molecules that inhibit MASP-2 biological activity (such as small molecule inhibitors, anti-MASP-2 antibodies, or blocking peptides that interact with MASP-2 or interfere with a protein-protein interaction) and molecules that decrease MASP-2 expression (such as MASP-2 antisense nucleic acid molecules, MASP-2 specific RNAi molecules and MASP-2 eribozymes), preventing this MASP-2 mode of activating alternate complement paths. MASP-2 inhibitory agents may be used alone as a primary therapy or in combination with other therapeutic products as an adjuvant therapy to enhance the therapeutic benefits of other medical treatments.

Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in a complement system component that occurs as a result of the administration of a MASP-2 inhibitory agent according to the methods of the invention: generation or production of MASP-2 dependent complement system activation products C4b, C3a, C5a and / or C5b-9 (MAC) (measured, for example, as described in Example 2), the reduction of alternative complement activation evaluated in a hemolytic assay using unsensitized red rabbit or guinea pig blood cells, reducing C4 cleavage and C4b deposition (measured, for example as described in Example 2) or reducing C3 cleavage and C3b deposition (measured, as described in Example 2). According to the present invention, the inhibitors of MASP-2 are used which are effective in inhibiting activation of the system. MASP-2 dependent decomposition. MASP-2 inhibiting agents useful in practicing this aspect of the invention include, for example, anti-MASP-2 antibodies and fragments thereof, MASP-2 inhibiting peptides, small molecules, soluble receptors and MASP-2 expression inhibitors. MASP-2 inhibitory agents may inhibit activation of the MASP-2 dependent decomposition system by blocking the biological function of MASP-2. For example, an inhibitory agent can effectively block protein-to-protein interactions of MASP-2, interfere with MASP-2 dimerization or assembly, block Ca binding, interfere with the MASP-2 serine protease site, or can reduce MASP-2 protein expression.

In some embodiments, deMASP-2 inhibitory agents that selectively inhibit MASP-2 complement activation, leaving Clq functionally dependent complement activation intact.

In one embodiment, a MASP-2 inhibitory agent useful in the methods of the invention is a specific MASP-2 inhibitory agent that specifically binds to a polypeptide comprising SEQ ID NO: 6 with an affinity of at least 10 times greater than for other antigens in the complement system. In another embodiment, a MASP-2 inhibitor specifically binds to a polypeptide comprising SEQ ID NO: 6 with a binding affinity of at least 100 times greater than for other antigens in the complement system. The binding affinity of the MASP-2 inhibiting agent may be determined using a suitable binding assay.

The MASP-2 polypeptide exhibits a molecular structure similar to MASP-1, MASP-3 and Clr and Cls, the protease cleavage system proteases. The cDNA molecule shown in SEQ ID NO: 4 encodes a representative example of MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO: 5) and provides the human deMASP-2 polypeptide with a leader sequence (amino acid 1 to 15 ) which is cleaved after secretion, resulting in the mature form of human MASP-2 (SEQ ID NO: 6). As shown in FIGURE 2, the human MASP 2 gene encompasses twelve exons. Human MASP-2 cDNA is encoded by exons B, C, D, F, G, Η, I, J, K, and L. An alternative junction results in a 20 kDa protein called MBL 19-associated protein ("MApl9"). ", also referred to as" sMAP ") (SEQ ID NO: 2), encoded by (SEQ ID NO: 1) seorigin from exons B, C, D and E as shown in FIGURE 2. The decDNA molecule shown in SEQ ID NO : 50 encodes murine MASP-2 (which consists of the amino acid sequence shown in SEQ ID NO: 51) and provides the murine MASP-2 polypeptide with a leader sequence that is cleaved after secretion, resulting in the mature form of demurine MASP-2 (SEQ ID NO: 52). The cDNA molecule set forth in SEQ ID NO: 53 encodes rat MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO: 54) and provides the rat MASP-2 polypeptide with a leader sequence which is cleaved after secretion, resulting in the mature form of rat MASP-2 (SEQ ID NO: 55).

Those skilled in the art recognize that the sequences disclosed in SEQ ID NO: 4, SEQ ID NO: 50 and SEQ ID NO: 53 represent unique human, murine and rat MASP-2 rings respectively and that alternative allelic variation and junction is expected to occur. . The allelic variants of the nucleotide sequences shown in SEQ ID NO: 4, SEQ IDNO: 50 and SEQ ID NO: 53, including those containing silent mutations and those where mutations result in amino acid sequence changes, are within the scope of the present invention. Allelic variants of the MASP-2 sequence can be cloned by probing cDNA or genomic libraries of different individuals according to standard procedures.

The domains of the human MASP-2 protein (SEQ ID NO: 6) are shown in FIGURE 3A and include an N-terminal sea urchin / Clr / Cls / Vegf protein-forming domain (CUBI) (amino acids of 1 SEQ ID NO: 6), an epidermal growth factor equivalent domain (amino acids 122 to 166), a second CUBI domain (167 to 293 amino acids), as well as a complement control protein domain tandem and a of serine protease. Alternative junctions of the MASP 2 gene result in MAp 19 shown in FIGURE 3B. MAp 19 is a non-enzymatic protein containing the MASP-2 N-terminal CUB1-EGF region with four additional residues (EQSL) derived from exon E as shown in FIGURE 2.

Several proteins have been shown to bind to or interact with MASP-2 through protein to protein interactions. For example, MASP-2 is known to bind to and form Cacom-dependent complexes, the lectin MBL proteins, H-ficoline and L-ficoline. Each MASP-2 / lectin complex has been shown to activate complement by dependent MASP-2 cleavage of C4 and C2 proteins (Ikeda, K., et al., J. Biol. Chem. 2662: 7451-7454, 1987; Matsushita, M et al., J. Exp. Med. 176: 1497-2284,2000 (Matsushita, M., et al., J. Immunol. 168: 3502-3506, 2002). Studies have shown that MASP-2 CUB1-EGF domains are essential for associating MASP-2 with MBL (Thielens, N.M., et al., J. Immunol. 166: 5068, 2001). CUB1EGFCUBII domains have also been shown to mediate MASP-2 dimerization, which is required for the formation of an active MBL complex (Wallis, R., et al., J. Biol. Chem. 275: 30962-30969,2000). Therefore, MASP-2 inhibitory agents can be identified that bind to or interfere with MASP-2 target regions known to be important for MASP-2 dependent complement activation. ANTI-MASP-2 ANTIBODIES

In some embodiments of this aspect of the invention, the MASP-2 inhibitor agent comprises an anti-MASP-2 antibody that inhibits activation of the MASP-2 dependent complement system. Anti-MASP-2 antibodies useful in this aspect of the invention include polyclonal, monoclonal or recombinant antibodies derived from any antibody producing mammal and may be multispecific, chimeric, humanized, anti-idiotypic and antibody fragments. Antibody fragments include Fab, Fab ', F (ab) 2, F (ab') 2, Fv fragments, scFv fragments and single decade antibodies as further described herein.

Several anti-MASP-2 antibodies have been described in the literature, some of which are listed below in TABLE 1. These anti-MASP-2 antibodies described above can be screened for their ability to inhibit activation of the MASP-2 dependent complement system using the assays herein. described. For example, MASP-2 Fab2 antirate antibodies have been identified that block MASP-2 dependent complement activation, as described in more detail in Examples 24 and 25 here. Since an anti-MASP-2 antibody is identified to function as a MASP-2 inhibiting agent, it can also be used to produce anti-idiotypic antibodies and used to identify other MASP-2 binding molecules as described below.

Table 1: MASP-2 specific antibodies from the literature

<table> table see original document page 114 </column> </row> <table> <table> table see original document page 115 </column> </row> <table>

ANTI-MASP-2S ANTIBODY WITH REDUCED EFFECTOR FUNCTION

In some embodiments of this aspect of the invention, anti-MASP-2 antibodies have reduced effector function in order to reduce the inflammation that may arise from activation of the classical complement pathway.

The ability of IgG molecules to trigger the complement-classical pathway has been shown to reside within the Fc portion of the molecule (Duncan, A. R., et al., Nature 332: 738-740 1988). IgG molecules in which the Fc portion of the molecule has been removed by enzymatic cleavage are devoid of this effector function (see Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Consequently, antibodies with reduced effector function may be generated as a result of the lack of the Fc portion of the molecule by having a genetically engineered Fc sequence that minimizes effector function or being from human IgG2 or IgG4 isotypes. Antibodies with reduced effector function may be produced by biological manipulation. molecular weight of the Fc portion of the IgG heavy chains as described in Example 9 herein and also described in Jolliffe, et al., Intl Rev. Immunol. 10: 241-250, 11993 and Rodrigues, et al., 5 J. Immunol. 151: 6954-6961, 1998. Antibodies with reduced effector function also include human IgG2 and IgG4 isotypes that have a reduced ability to activate complement and / or interact with Fc receptors (Ravetch, JV, et al., Annu. Rev. Immunol. 9 : 457-492, 1991; Isaacs, JD, et al., J. Immunol. 148: 3062-3071, 1992; van de Winkel, JG, et al., Immunol. Today 14: 215-221, 1993). Humanly fully humanized antibodies specific for human MASP-2 comprised of IgG2 or IgG4 isotypes may be produced by one of several methods known to one of ordinary skill in the art, as described in Vaughan, TJ, et al., Nature Biotechnical 16: 535-539 , 1998.

ANTI-MASP-2S ANTIBODY PRODUCTION

Anti-MASP-2 antibodies can be produced using MASP-2 polypeptides (e.g., life-size MASP-2) or by using peptides carrying antigenic MASP-2 epitope (e.g., a portion of the MASP-2 polypeptide). Immunogenic peptides can be as small as five amino acid residues. For example, MASP-2 opolipeptide including the entire amino acid sequence of SEQ IDNO: 6 may be used to induce anti-MASP-2 antibodies useful in the method of the invention. Particular MASP-2 domains known to be involved in protein-protein interactions, such as the CUBI and CUBIEGF domains, as well as the region encompassing the active serine protease site, may be expressed as recombinant polypeptides as described in Example 5 and used as antigens. In addition, peptides comprising a portion of at least 6 amino acids of the MASP-2 polypeptide (SEQ ID NO: 6) are also useful for inducing MASP-2 antibodies. Additional examples of MASP-2 derived antigens useful for inducing MASP-2 antibodies are TABLE 2. MASP-2 epolipeptide peptides used to elicit antibodies can be isolated as natural polypeptides or recombinant or synthetic catalytically inactive recombinant epolipeptides, such as MASP-2A, as further described in Examples 5 to 7. In some forms of In this aspect of the invention, anti-MASP-2 antibodies are obtained using a transgenic mouse coat as described in Examples 8 and 9 and described below.

Antigens useful for making anti-MASP-2 antibodies also include fusion polypeptides, such as fusions of MASP-2 or a portion thereof with an immunoglobulin polypeptide or maltose deletion protein. The polypeptide immunogen may be a life-sized molecule or a portion thereof. If the polypeptide moiety is equivalent to hapten, such moiety may be advantageously joined or linked to a macromolecular carrier (such as keyhole limb hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

Table 2: MASP-2 Derived Antigens

<table> table see original document page 117 </column> </row> <table> <table> table see original document page 118 </column> </row> <table>

Polyclonal Antibodies

Polyclonal MASP-2 antibodies can be prepared by immunizing an animal with MASP-2 polypeptide or an immunogenic portion thereof using methods well known to those of ordinary skill in the art. See, For example, Green, et al., "Production of Polyclonal Antisera," in Immunochemical Protocols (Manson, ed.), Page 105e as further described in Example 6. The immunogenicity of a MASP-2 polypeptide may be enhanced by the use of an adjuvant, including mineral gels such as aluminum hydroxide or Freund's adjuvant (complete or incomplete), surface active substances such as lysolecithin, polyolspluronics, polyanions, oily emulsions, keyhole limpet hemocyanin and dinitrophenol. Polyclonal antibodies are typically raised in animals such as horses, cows, dogs, chickens, rats, mice, rabbits, guinea pigs, goats or sheep. Alternatively, an anti-MASP-2 antibody useful in the present invention may also be derived from a subhuman primate. General techniques for inciting diagnostically and therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication No. WO91 / 11465 and in Losman, MJ, et al., Int. J. Cancer 46: 310, 1990. Seroscontaining immunologically active antibodies are then produced from the blood of such immunized animals using standard procedures well known in the art. MONOCLONAL ANTIBODIES

In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody. Anti-MASP-2 monoclonal antibodies are highly specific and are directed against a unique MASP-2 epitope. As used herein, the "monoclonal" modifier indicates the antibody character as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring antibody production by any particular method. Monoclonal antibodies may be obtained using any technique that provides for the production of antibody molecules by continuous cell lines in culture, such as the hybridoma method described by Kohler, G., et al., Nature 256: 495, 1975 or they may be manufactured. by the methods of recombinant DNA (see, for example, U.S. Patent No. 4,816,567 issued to Cabilly). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson, T., et al., Nature 352: 624-628, 1991 and Marks, J. D., et al., J. Mol.Biol. 222: 581-597 (1991). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclasses.

For example, monoclonal antibodies may be obtained by injection into a suitable mammal (e.g., a mouse BAB / c) with a composition comprising a MASP-2 polypeptide or portion thereof. After a predetermined period of time, splenocytes are removed from the mouse and suspended in a cell culture medium. Splenocytes are then fused to an immortal cell line to form a hybridoma. the hybridomasformed are cultured in cell culture and screened for their ability to produce a monoclonal antibody against MASP-2. An example further describing the production of anti-MASP-2 monoclonal antibodies is provided in Example 7. (See also Current Protocols in Immunology, Vol. 1, John Wiley & Sons, pages 2.5.1-2.6.7, 1991).

Human monoclonal antibodies can be obtained through the use of transgenic mice that have been engineered to produce specific human antibodies in response to antigenic inoculation. In this technique, elements of the human immunoglobulin heavy and light chain sites are introduced into mouse strains derived from embryonic stem cells. which contain targeted interruptions of the endogenous immunoglobulin heavy chain and light chain sites. Transgenic mice can synthesize human antibodies specific for human antigens such as the MASP-2 antigens described herein and mice can be used to produce human MASP-2 antibody-secreting hybridomas by fusing B-cells from such animals to myeloma cell lines. suitable using conventional Kohler-Milstein technology as further described in Example 7. Transgenic mice with a human immunoglobulin genome are commercially available (e.g., from Abgenix, Inc., Fremont, CA and Medarex, Inc., Annandale, NJ). Methods for obtaining human antibodies from transgenic mice are described, for example, by Green, L. L., et al., Nature Genet. 7: 13, 1994; Lonberg, N., et al., Nature 368: 856, 1994; and Tailor, L. D., et al., Int. Immun. 6: 579, 1994.

Monoclonal antibodies may be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include Protein-A Sepharose affinity chromatography, size exclusion chromatography and ion exchange chromatography (see, for example, Coligan on pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G (IgG)," in Methods in Molecular Biology, The Human Press, Inc., Vol. 10, pages 79-104, 1992).

Once produced, polyclonal, monoclonal, or phage-derived antibodies are first tested for specific MASP-2 binding. A variety of assays known to those skilled in the art can be used to detect antibodies that specifically select MASP-2. Exemplary assays include Western blot analysis or immunoprecipitation by standard methods (e.g., as described in Ausubel et al), immunoelectrophoresis, enzyme linked immunosorbent assays, dot blots, inhibition or competition assays, and interleaved assays (as described in Harlow and Land, Antibodies : A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). Since antibodies are specifically identified to bind MASP-2, anti-MASP-2 antibodies are tested for their ability to function as a DEMASP-2 inhibitory agent in one of several assays such as, for example, a slope-down assay. Lectin specific C4 (described in Example 2), a C3b deposition assay (described in Example 2) or a C4b deposition assay (described in Example 2).

The affinity of anti-MASP-2 monoclonal antibodies can be easily determined by one of ordinary skill in the art (see, for example, Scatchard, A., NY Acad. Sci. 51: 660-672, 1949). In one embodiment, anti-MASP-2 monoclonal antibodies useful for the methods of the invention bind to MASP-2 with a binding affinity of <100 nM, preferably <10 nM and most preferably <2 nM.

CHEMICAL / HUMANIZED ANTIBODIES

Monoclonal antibodies useful in the method of the invention include chimeric antibodies in which a portion of the heavy and / or light chain is identical with or homologous to the corresponding antibody sequences derived from a particular species or belonging to a particular antibody class or subclass, while the rest of ) chain (s) is identical with or homolog to the corresponding antibody sequences derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies (US Patent No. 4,816,567 issued to Cabilly and Morrison, SL, et. al., Proc. Nat'l Acad. Sci. USA 81: 6851-6855, 1984).

One form of a chimeric antibody useful in the invention is a humanized anti-MASP-2 monoclonal antibody. Humanized forms of non-human (e.g. murine) antibodies are chimeric antibodies, which contain minimal sequences derived from non-human immunoglobulin. Humanized monoclonal antibodies are produced by transferring non-human complementarity determining regions (CDRs) (for example, mouse). ) of the mouse daimmunoglobulin heavy and light variable chains in a human variable domain. Typically, human antibody residues are then replaced in the matrix regions of non-human counterparts. In addition, humanized antibodies may comprise residues that are not found in the receptor antibody or donor antibody. These modifications are made to further degrade antibody performance. In general, the humanized antibody will comprise substantially all of at least one typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the Fv matrix regions are those of a human immunoglobulin sequence. The optionally humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see Jones, P. T., et al., Nature 321: 522-525, 1986; Reichmann, L., et al., Nature 332: 323-329, 1988; ePresta, Curr. Op. Struct. Biol. 2: 593-596, 1992. Humanized antibodies useful in the invention include human monoclonal antibodies including at least one CDP3 deletion region of MASP-2. In addition, the Fc moieties may be substituted to produce IgA or IgM antibodies as well as human IgG. Such humanized antibodies will have particular clinical utility because they will specifically recognize human MASP-2 but will not evoke a human immune response against the antibody itself. Consequently, they are best suited for in vivo administration to humans, especially when repeated or long-term administration is required.

An example of generating a humanized anti-MASP-2 antibody from a murine monoclonal anti-MASP-2 antibody is given here in Example 10. Techniques for producing humanized monoclonal antibodies are also described, for example, by Jones, PT , et al., Nature 321: 522, 1986; Carter, P., et al., Proc. Nat'l. Acad. Sci. USA 89: 4285, 1992; Sandhu, J. S., Crit. Rev. Biotech. 12: 437, 1992; Singer, I.I., Et al., J. Immun. 150: 2844, 1993; Sudhir (ed.), Engineering Protocols Antibody, Humana Press, Inc., 1995; Kelley, "Engineering Therapeutic Antibodies," in Protein Engineering: Principles and Practice, Cleland et al. (Eds.), John Wiley & Sons, Inc., pages 399-434, 1996; and U.S. Patent No. 5,693,762 issued to Queen, 1997. In addition, there are commercial entities that will synthesize humanized antibodies from specific murine antibody regions such as Protein Design Labs (MountainView, CA).

RJE C QMBIN A NTE S ANTIBODY

Anti-MASP-2 antibodies may also be made using recombinant methods. For example, human antibodies may be made using human immunoglobulin expression libraries (available for example from Stratagene, Corp., La Jolla, CA) to produce human antibody fragments (VH, VL, Fv5 Fd, Fab, or F (ab ')2). These fragments are then used to construct whole human antibodies using techniques similar to those for producing chimeric antibodies.

ANTI-IDIOTIC Antibodies

Since anti-MASP-2 antibodies are identified with the desired inhibitory activity, these antibodies can be used to arrest anti-idiotypic antibodies that resemble a portion of MASP-2 using techniques that are well known in the art. See, for example, Greenspan, NS, et al., FASEB J. 7: 437, 1993. For example, antibodies that bind MASP-2 and competitively inhibit a deMASP-2 protein interaction required for complement activation may anti-idiotypes that resemble the MBL binding site in the MASP-2 protein and therefore bind and neutralize a deMASP-2 binding ligand such as, for example, MBL.

IMMUNOGLOBULIN FRAGMENTS

MASP-2 inhibiting agents useful in the method of the invention encompass not only intact immunoglobulin molecules but also well-known fragments including Fab, Fab ', F (ab) 2, F (ab') 2 eFv, scFv, diabodies, linear antibodies fragments. , single chain antibody molecules and multispecific antibodies formed from antibody fragments.

It is well known in the art that only a small portion of an antibody molecule, the paratope, is involved in binding the antibody to its epitope (see, for example, Clark, WR, The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., NY , 1986). The pFc 'and Fc regions of the antibody are effectors of the complement classic pathway, but are not involved in antigen binding. An antibody from which the pFc 'region has been enzymatically cleaved or produced in the pFc' region is designated an F (ab ') 2 fragment and retains both antigen binding sites of an intact antibody. An isolated F (ab ') 2 fragment is alluded to as a bivalent monoclonal fragment because of the two antigen binding sites. Similarly, an antibody from which the Fc region was enzymatically cleaved or which was produced without the Fc region is called a Fab fragment and retains one of the antigen binding sites of an intact antibody molecule.

Antibody fragments may be obtained by proteolytic hydrolysis, such as by pepsin or papain digestion of antibody by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of pepsin antibodies to provide a 5S fragment denoted F (ab ') 2. This fragment may be further cleaved using a thiol reducing agent to produce 3.5S Fab 'monovalent fragments. Optionally, the cleavage reaction may be performed using a blocking group for sulfhydryl groups resulting from cleavage of disulfide bonds. As an alternative, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and one Fc fragment directly. These methods are described, for example, U.S. Patent. No. 4,331,647 issued to Goldenberg; Nisonoff, A., et al., Arch.Biochem. Biophys. 89: 230, 1960; Porter, R.R., Biochem. J. 73: 119, 1959, Edelman, et al., In Methods in Enzymology, 1: 422, Academic Press, 1967; e by Coligan on pages 2.8.1-2.8.10 and 2.10.-2.10.4.

In some embodiments, the use of antibody fragments lacking the Fc region is preferred to prevent activation of the classical complement pathway that is initiated on Fc binding to the Fc receptor. There are several methods by which a MoAb can be produced that avoids Fcy receptor interactions. For example, the Fc region of an anti-clonoclonal may be chemically removed using partial digestion by proteolytic enzymes (such as phycin digestion), thereby generating, for example, antigen-binding antibody fragments such as Fab or F (ab) 2 fragments. (Mariani, M., et al., Mol. Immunol. 28: 69-71,1991).

Alternatively, the human γ4 IgG isotype, which does not bind Fcy receptors, may be used during the construction of a humanized antibody as described herein. Antibodies, single chain antibodies, and antigen binding domains lacking the Fc domain can also be engineered using recombinant techniques described herein.

SINGLE CHAIN ANTIBODY FRAGMENTS

Alternatively, MASP-2 specific single peptide chain binding molecules can be created in which the heavy and light chain Fv regions are attached. The Fv fragments may be attached by a peptide linker to form a single chain antigen binding protein (scFv). These single stranded antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the Vh and VL domains that are connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell, such as E. coli. Recombinant host cells synthesize a single polypeptide chain with a linker peptide that bridges the two V domains. scFvs production are described for example by Whitlow, et al., Methods: A Companion to Methods in Enzymology 2: 97, 1991; Bird, et al., Science 242: 423, 1988; U.S. Patent No. 4,946,778 issued to Ladner; Pack, P., et al., Bio / Technology 11: 1271, 1993.

As an illustrative example, a MASP-2-specific scFv may be obtained by exposing lymphocytes to the invitro MASP-2 polypeptide and selecting antibody demonstration libraries on the phage or similar vectors (for example, by using the DEMASP-2 protein or peptide). 2 fixed or labeled). Genes encoding polypeptides having potential MASP-2 polypeptide binding domains can be obtained by screening for random peptide libraries demonstrated in phage or in bacteria such as E. coli. These random peptide demonstration libraries can be used to screen for peptides that interact with MASP-2 techniques to create and screen such random peptide demonstration libraries are well known in the art (U.S. Patent No. 5,223,409 issued to Lardner; Patent U.S. Patent No. 4,946,778 issued to Ladner; US Patent No. 5,403,484 issued to Lardner; US Patent No. 55,571,698 issued to Lardner; and Kay et al., Phage Display of Peptides and Proteins Academic Press, Inc., 1996) and libraries of Random peptide demonstration and screening kits for such libraries are commercially available, for example, from CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass. .) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ.).

Another form of an anti-MASP-2 antibody fragment useful in this aspect of the invention is a peptide encoding a unique complementarity determining region (CDR) that binds to an epitope on a MASP-2 antigen and inhibits MASP-dependent complement activation. -2. CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding CDRs of an antibody of interest. Such genes are prepared, for example, by using polymerase chain splicing to synthesize the variable region of antibody-producing RNA (see, for example, Larrick et al., Methods: Accompaniment to Methods in Enzymology 2: 106, 1991 Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (Eds.), Page 166, Cambridge University Press, 1995; and Ward et al., "GeneticManipulation and Expression of Antibodies, "in Monoclonal Antibodies: Principles and Applications, Birch et al. (Eds.), Page 137, Wiley-Liss, Inc., 1995).

The MASP-2 antibodies described herein are administered to a patient in need thereof to inhibit MASP-2 dependent complement activation. In some embodiments, the MASP-2 inhibitory agent is a high affinity human or humanized anti-MASP-2 monoclonal antibody with reduced effector function.

PEPTIDE INHIBITORS

In some embodiments of this aspect of the invention, the MASP-2 inhibitor agent comprises isolated MASP-2 peptide inhibitors, including isolated natural peptide inhibitors and synthetic depeptide inhibitors that inhibit activation of the MASP-2 complement-dependent system. As used herein, the term "isolated MASP-2 peptide inhibitors" refers to peptides that inhibit MASP-2-dependent complement-activation activation by binding to, competing with MASP-2 for binding to another recognition molecule (e.g. MBL, H-ficoline, M-ficoline or L-ficoline) in the lectin pathway, and / or directly interacting with MASP-2 to inhibit MASP-2-dependent complement activation which are substantially pure and are essentially free of other substances with that they can be found nanature to a practical and appropriate degree as to their intended use.

Peptide inhibitors have been successfully used in vivo to interfere with protein-protein interactions and catalytic sites. For example, peptide inhibitors for structurally related LFA-I adhesion molecules have recently been approved for clinical use in coagulopathies (Ohman, E.M., et al., European Heart J. 16: 50-55, 1995). Short linear peptides (<30 amino acids) have been described that prevent or interfere with integrin-dependent adhesion (Murayama, O., et al., J. Biochem. 120: 445-51, 1996). Longer peptides, ranging in length from 25 to 200 amino acid residues, have also been successfully used to block integrin-dependent adhesion (Zhang, L., et al., J. Biol. Chem. 271 (47): 29953-57 , 1996). Generally, longer peptide inhibitors have higher affinities and / or slower dissociation indices than short peptides and therefore may be more potent inhibitors. Cyclic peptide inhibitors have also been shown to be effective inhibitors of integrins in vivo for the treatment of human inflammatory disease (Jackson, D. Y., et al., J. Med. Chem. 40: 3359-68, 1997). One method of producing cyclic peptides involves the synthesis of peptides wherein the terminal amino acids of the peptide are cysteines, thereby allowing the peptide to exist in a cyclic form of disulfide bonding between terminal amino acids, which have been shown to improve affinity and half life in vivo for the treatment of hematopoietic neoplasms (e.g., US Patent No. 6,649,592 issued to Larson).

SYNTHETIC MASP-2 PEPTIDE INHIBITORS

MASP-2 inhibitor peptides useful in the methods of this invention are exemplified by amino acid sequences that burn out target regions important for MASP-2 function. Peptide inhibitors useful in practicing the methods of the invention range in size from about 5 amino acids to about 300 amino acids. TABLE 3 provides a list of exemplary inhibitor peptides that may be useful in practicing this aspect of the present invention. A MASP-2 inhibitor peptide candidate can be tested for ability to function as a MASP-2 inhibitor in one of several assays including, for example, a lectin-specific C4 cleavage assay (described in Example 2) and a deposition assay of C3b (described in Example 2).

In some embodiments, the MASP-2 inhibitor peptides are derived from MASP-2 polypeptides and are selected from mature and life-size MASP-2 protein (SEQ ID NO: 6) or a particular MASP-2 protein domain such as such as the CUBI domain (SEQ ID NO: 8), the CUBIEGF domain (SEQ ID NO: 9), the EGF domain (SEQ ID NO: 11), and the serine protease domain (SEQ ID NO: 12). previously described, CUBEGFCUBII regions have been shown to be required for MBL dimerization and binding (Thielens et al., supra). In particular, the TFRSDYN peptide sequence (SEQ ED NO: 16) in the MASP-2 CUBI domain was shown to be involved in binding to MBL in a study that identified a human carrying a homozygous mutation from Asp 105 to Gly 105, resulting in loss of MASP-2 from the MBL complex (Stengaard-Pedersen, K., et al., New England J. Med. 349: 554-560, 2003).

MASP-2 inhibitor peptides may also be derived from MApl9 (SEQ ID NO: 3). As described in Example 30, MAp 19 (SEQ ID NO: 3) (also referred to as sMAP), has the ability to downregulate the lectin pathway, which is activated by the MBL complex. Iwaki et al., J. Immunol. 177: 8626-8632, 2006. Although not wishing to be bound by theory, it is likely that sMAP will be able to occupy the MASP-2 / sMAP deletion site in MBL and prevent MASP-2 from binding to MBL. It has also been reported that sMAP competes with MASP-2 in combination withficolin A and inhibits complement activation by the ficolinA / MASP-2 complex. Endo Y. et al., Immunogenetics 57: 837-844 (2005).

In some embodiments, the MASP-2 inhibitor peptides are derived from MASP-2 binding lectin proteins and are involved in the lectin complement pathway. Several different lectins have been identified that are involved in this pathway, including mannan-binding (MBL) -olectin, L-ficoline, M-ficoline and H-ficoline. (Ikeda, K., et al., J. Biol. Chem. 262: 7451-7454, 1987; Matsushita, M., et al., J. Exp. Med. 176: 1497-2284, 2000; Matsushita, M. , et al., J. Immunol. 168: 3502-3506, 2002). These lectins are present in serum as homotrimeric disubunit oligomers, each having N-terminal collagen equivalent fibers with carbohydrate recognition domains. These different lectins have been shown to bind MASP-2 and the lectin / MASP-2 complex activates complement by cleavage of C4 and C2 proteins. H-pholinolin has a 24 amino acid amino terminal region, a collagen-equivalent domain with 11 Gly-Xaa-Yaa repeats, a 12-amino acid bottleneck domain, and a fibrinogen equivalent domain of 207 amino acids (Matsushita, M., et al., J Immunol 168: 3502-3506, 2002). H-phytoline binds to GlcNAc and agglutin-coated human erythrocytes coated with S. typhimurium, S. minnesota and E. coli. H-pholine has been shown to be associated with MASP-2 and MAp 19 and activates the lectin pathway. Id. L-ficoline / P35 also binds GlcNAc and has been shown to be associated with MASP-2 and MAp 19 in human serum and this complex has been shown to activate the lectin pathway (Matsushita, M., et al., J. Immunol. 164 : 2281, 2000). Accordingly, MASP-2 inhibitor peptides useful in the present invention may comprise a region of at least 5 amino acids selected from the MBL protein (SEQ ID NO: 21), the H-ficoline protein (Genbank accession number NM 173452), the M protein. -pholine (Genbank accession number 000602) and L-Ficoline protein (Genbank accession number NM 015838).

More specifically, scientists have identified the MASP-2 deletion site in MBL as being within the 12 triplets Gly-XY "GKD GRD GTK GEK GEP GQG LQG POG KLG POG NOG PSGSOG PKG QKG DOG KS" (SEQ ID NO: 26) residing between the hinge and the neck at the C-terminal portion of the MBP collagen equivalent domain (Wallis, R., et al., J. Biol. Chem. 279: 14065, 2004). This MASP-2 binding site region is also highly conserved in human H-ficoline and human L-ficoline. A consensus binding site has been described that is present on all three lectin proteins comprising the amino acid sequence "OGK-X-GP" (SEQ ID NO: 22) where the letter "O" represents hydroxyproline and the letter "X" is a hydrophobic residue (Wallis et al., 2004, supra). Accordingly, in some embodiments, MASP-2 inhibitor peptides useful in this aspect of the invention are at least 6 amino acids in length and comprise SEQ ID NO: 22. MBL-derived peptides that include the amino acid sequence "GLRGLQ GPO GKL GPO G "(SEQ ID NO: 24) has been shown to bind invitro MASP-2 (Wallis, et al., 2004, supra). To enhance binding to MASP-2, peptides can be synthesized that are flanked by two GPO triplets at each end ("GPO GPO GLR GQ GPO GKG OGP O" SEQ IDNO: 25 to enhance triple helix formation as found in the native MBL protein (as further described in Wallis, R., et al., J. Biol. Chem. 279: 14065, 2004).

MASP-2 inhibitor peptides may also be derived from human H-pholine which include the sequence "GAO GSO GEKGAO GPQ GPO GPO GKM GPK GEO GDO" (SEQ ID NO: 27) from the H-consensus MASP-2 binding region ficolina. Also included are peptides derived from human L-ficoline which include the sequence "GCOGLO GAO GDK GEA GTN GKR GER GPO GPO GPO GPN GAOGEO" (SEQ ID NO: 28) from the consensus MASP-2 binding region in L-ficoline.

MASP-2 inhibitor peptides may also be derived from the C4 cleavage site such as "LQRALEILPNRVTIKANRPFLVFI" (SEQ ID NO: 29) which is the C4 declining site linked to the C-terminal portion of antithrombin III (Glover, GI, etal., Mol. Immunol 25: 1261 (1988)) Table 3: Exemplary MASP-2 Inhibitor Peptides

<table> table see original document page 133 </column> </row> <table>

Note: The letter "O" represents hydroxyproline. The letter "X" is a hydrophobic residue. Peptides derived from the C4 cleavage site as well as other peptides that inhibit the MASP-2 serine protease site can be chemically modified to be reversible protease inhibitors. For example, appropriate modifications may include, but are not necessarily limited to, halomethyl ketones (Br, Cl, I, F) noterminal C, Asp or Glu, or attached to functional side chains; haloacetyl (or other α-haloacetyl) groups on amino groups or other functional side chains; epoxide or imine containing groups at the amino or carboxy termini or functional side chains; or imidate esters on the amino or carboxy terminals or functional side chains. Such modifications would provide the advantage of permanently inhibiting the enzyme by covalently binding the peptide. This may result in lower effective doses and / or a need for less frequent administration of the inhibitor peptide.

In addition to the inhibitor peptides described above, MASP-2 inhibitor peptides useful in the method of the invention include peptides containing the anti-MASP-2 moAb MASP-2 binding region CDR3 obtained as described herein. The sequence of CDR regions for peptide synthesis can be determined by known methods. The heavy chain variable region is a peptide that generally ranges from 100 to 150 amino acids in length. The light chain variable region is a peptide that generally ranges from 80 to 130 amino acids in length. CDR sequences within the heavy and light chain variable regions include only about 3 to 25 amino acid sequences that can be easily sequenced by a person of common natures ability.

Those skilled in the art will recognize that substantially homologous variations of the above described MASP-2 inhibitor peptides will also exhibit MASP-2 inhibitory activity. Exemplary variations include, but are not necessarily limited to, peptide having additional insertions, deletions, substitutions and / or amino acids in the carboxy or amino terminal portions of the peptide objects thereof. Accordingly, those homologous peptides having MASP-2 inhibitory activity are considered to be useful in the methods of this invention. The described peptides may also include duplicate motifs and other modifications with conservative substitutions. Conservative variants are described elsewhere herein and include changing one amino acid by another of similar charge, size or hydrophobicity and the like.

MASP-2 inhibitor peptides may be modified to increase solubility and / or to maximize positive or negative charge to more closely resemble the segment in the protein protein. The derivative may or may not have the primary amino acid structure of exactly one peptide disclosed herein as long as the derivative functionally has the desired inhibition property of MASP-2. Modifications may include amino acid substitution with one of the twenty commonly known amino acids or another amino acid, a derivatized or substituted amino acid with desirable ancillary characteristics, such as resistance to enzymatic degradation or a D-amino acid or substitution with another molecule or compound, such as a carbohydrate, which mimics the natural confirmation and function of the amino acid, amino acids or peptide, amino acid deletion; amino acid insertion with one of the commonly known twenty-amino acids or another amino acid, common amino acid derivatized or substituted with desirable ancillary characteristics, such as resistance to enzymatic degradation or a D-amino acid or substitution with another molecule or compound, such as a carbohydrate, which mimic the natural confirmation and function of the amino acid, amino acids or peptide; or substitution with another molecule or compound, such as a carbohydrate or nucleic acid monomer, burns the natural conformation, charge distribution, and function of the peptide precursor. Peptides may also be modified by acetylation or amidation.

Synthesis of derived inhibitor peptides may rely on known techniques of peptide biosynthesis, carbohydrate biosynthesis and others. As a starting point, the technician can rely on a suitable computer program to determine the conformation of a peptide of interest. Once the peptide conformation disclosed herein is known, then the skilled artisan can determine in a rational design manner what kind of substitutions can be made at one or more sites to frame a derivative that retains the basic conformation and carries the distribution of the precursor peptide but can possess characteristics that are not present or enhanced in relation to those found in the precursor peptide. Once candidate derivative molecules are identified, the derivatives can be tested to determine whether they function as MASP-2 inhibitory agents using the above-described assays.

SCREENING FOR MASP-2 INHIBITOR PEPTIDES

Molecular modeling and rational molecular planning can also be used to generate and screen for peptides that mimic the molecular structures of MASP-2 key binding regions and inhibit MASP-2 complement activities. Molecular structures used for modeling include CDR regions of monoclonal anti-MASP-2 antibodies, as well as target regions known to be important for MASP-2 function including the region required for polymerization, the region involved in binding to MBL and the active site of serinaprotease as previously described. Methods for identifying peptides that bind to a particular target are well known in the art, for example, molecular printing can be used to construct novel macromolecular structures such as peptides that bind to a particular molecule. See, for example, Shea, K. J., "Molecular Imprinting of Synthetic Network Polymers: The New Synthesis of Macromolecular Binding and Catalytic Sties," TRIP 2: (5) 1994.

As an illustrative example, a method of preparing MASP-2 binding peptide mimics is as follows. Functional monomers of a known MASP-2 binding peptide or the deletion region of an anti-MASP-2 antibody exhibiting inhibition of MASP-2 (standard) are polymerized. The pattern is then removed, followed by polymerization of a second class of monomers in the void left by the standard to provide a new molecule that exhibits one or more desired properties that are similar to the pattern. In addition to preparing peptides in this manner, other MASP-2 binding molecules that are MASP-2 inhibiting agents such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids and other biologically active materials may also be prepared. This method is useful for designing a wide variety of biological imitations that are more stable than their natural counterparts because they are typically prepared by free-radical polymerization of functional monomers, resulting in a compound with a non-biodegradable main chain.

SUMMARY OF PEPTIDE

MASP-2 inhibitor peptides may be prepared using techniques well known in the art, such as the synthetic solid phase technique initially described by Merrifield in J. Amer. Chem. Sound. 85: 2149-2154, 1963. Automated synthesis can be obtained, for example, using Applied Biosystems 43IA Peptide Synthesizer (Foster City, Calif.) According to the instructions provided by the manufacturer. Other techniques can be found, for example, in Bodanszky, M., et al., Peptide Synthesis, Second Edition, John Wiley & Sons, 1976, as well as in other reference works known to those skilled in the art.

Peptides may also be prepared using standard genetic engineering techniques known to those skilled in the art. For example, the peptide may be produced enzymatically by inserting nucleic acid encoding the peptide into an expression vector, expressing the DNA and translating the DNA into the peptide. in the presence of the required amino acids. The peptide is then purified using chromatographic or electrophoretic techniques or by means of a carrier protein that can be fused to and subsequently cleaved from the peptide by the expression noveltor insert in phase with the peptide coding sequence and nucleic acid sequence encoding the carrier protein. Protein-peptide fusion may be isolated using chromatographic, electrophoretic or immunological techniques (such as binding to a resin via an antibody to the protein). The peptide may be cleaved using chemical methodology or enzymatically, as for example by hydrolases.

MASP-2 inhibitor peptides which are useful in the invention method can also be produced in recombinant host cells following conventional techniques. To express a sequence encoding the MASP-2 inhibitor peptide, a nucleic acid molecule encoding the peptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then introduced into a host cell. In addition to transcriptional regulatory consequences, such as promoters and enhancers, expression vectors may include translational regulatory sequences and a marker gene, which are suitable for the selection of cells that carry expression vectors.

Nucleic acid molecules encoding a MASP-2 inhibitor peptide can be synthesized with "gene machines" using protocols such as the phosphoramidite method. If double-chemically synthesized stranded DNA is required for an application such as gene synthesis or gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 base pairs) is technically straightforward and can be accomplished by synthesizing the complementary filaments and then annealing them. For the production of longer genes, synthetic (double stranded) genes are assembled in modular form from double stranded fragments that are from 20 to 100 nucleotides in length. For reviews on cholinucleotide synthesis, see, for example, Glick and Pasternak, "Molecular Biotechnology, Principles and Applications of Recombinant DNA", ASMPress, 1994; Itakura, K., et al., Annu. Rev. Biochem. 53: 323, 1984; and Climie, S., et al., Proc. Nat'l Acad. Know. USA 87: 633, 1990.

SMALL MOLECULLE INHIBITORS

In some embodiments, the inhibitory agents of

MASP-2 are small molecule inhibitors including natural synthetic substances that have a low molecular weight, such as, for example, peptides, peptidomimetics and non-peptide inhibitors (including oligonucleotides and organic compounds). MASP-2 small molecule inhibitors can be generated based on the molecular structure of the variable regions of anti-MASP-2 antibodies.

Small molecule inhibitors can also be designed and generated based on the crystal structure of MASP-2 using computational drug planning (Kuntz I. D., et al., Science 257: 1078, 1992). The crystal structure of rat MASP-2 has been described (Feinberg, H., et al., EMBO J. 22: 2348-2359, 2003). Using the method described by Kuntz et al., The crystal lattice coordinates of MASP-2 are used as an input to a computer program such as DOCK, which produces a list of small molecule structures that are expected to bind to MASP-2. The use of such computer programs is well known to one of skill in the art, for example, the crystalline structure of the HTV-I protease inhibitor was used to identify non-peptide ligands that are HTV-I protease inhibitors by evaluating the adjustments of compounds found. in the Cambridge Crystallographic database for the enzyme binding site using the DOCK program (Kuntz, ID, et al., J. Mol. Biol. 161: 269-288, 1982; DesJarlais, RL, et al, PNAS 87: 6644-6648, 1990).

The list of small molecule structures that are identified by a computational method as potential MASP-2 inhibitors is screened using a MASP-2 binding assay as described in Example 7. Small molecules that are found to bind MASP-2 are then assayed in a functional assay as described in Example 2 to determine if they inhibit MASP-2-dependent complement activation.

Soluble MASP-2 RECEIVERS

Other suitable MASP-2 inhibiting agents are believed to include soluble MASP-2 receptors, which may be produced using techniques known to those of ordinary skill.

MASP-2 EXPRESSION INHIBITORS

In another embodiment of this aspect of the invention, the MASP-2 inhibiting agent is a MASP-2 expression inhibitor capable of inhibiting MASP-2 dependent complement activation. In the practice of this invention, representative MASP-2 expression inhibitors include antisense MASP-2 nucleic acid molecules (such as antisense comomRNA, antisense DNA or antisense oligonucleotides), MASP ribozymes -2 and MASP-2 RNAi molecules.

Antisense RNA and DNA molecules act to directly block MASP-2 mRNA transduction by MASP-2 aomRNA hybridization and prevent MASP-2 protein translation. An antisense nucleic acid molecule can be constructed in several different ways as long as it is capable of interfering with the expression of MASP-2. for example, an antisense nucleic acid molecule may be constructed by inverting the coding region (or a portion thereof) of the MASP-2 docDNA (SEQ ID NO: 4) from its normal transcription orientation to allow transcription of your complement.

The antisense nucleic acid molecule is modestly substantially identical to at least a portion of the target gene or genes. Nucleic acid, however, need not be perfectly identical to inhibit expression. In general, higher homology may be used to compensate for the use of a shorter antisense nucleic acid molecule. The minimum percentage identity is typically greater than about 65%, but a higher percentage identity may exert more effective repression of endogenous sequence expression. Substantially higher percent identity of more than about 80% is typically preferred, although about 95% up to absolute identity is typically more preferred.

The antisense nucleic acid molecule need not have the same intron or exon pattern as the target gene and non-coding segments of the target gene can be equally effective in achieving antisense suppression of target gene expression as segment coders. A DNA sequence of at least about another 8 nucleotides may be used as the antisense nucleic acid molecule, although a longer sequence is preferable. In the present invention, a representative example of a useful MASP-2 inhibitory agent is an antisense MASP-2 nucleic acid molecule that is at least ninety percent identical to the MASP-2 cDNA complement that consists of the presented nucleic acid sequence in SEQ ID NO: 4. Nucleic acid sequence shown in SEQ ID NO: 4 encodes the MASP-2 protein consisting of the amino acid sequence shown in SEQ IDNO: 5.

Targeting antisense oligonucleotides to bind MASP-2 mRNA is another mechanism that can be used to reduce the level of MASP-2 protein synthesis. For example, the synthesis of polygalacturonase and the muscarinic acetylcholine receptor type 2 is inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U.S. Patent No. 5,739,119 issued to U.S. Patent No. 5,759,829 issued to Shewmaker). In addition, examples of antisense inhibition have been demonstrated with antisense nuclear protein cyclin, multiple drug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAa receptor and human EGF (see, for example, US Patent No. 5,801,154 issued to Baracchini; US Patent No. 5,789,573 issued to Baker; US Patent No. 5,718,709 issued to Considine; and US Patent No. 5,610,288 issued to Reubenstein).

A system has been described which enables a person of ordinary skill to determine which oligonucleotides are useful in the invention, which involves probing for suitable sites on the target mRNA using Rnase H binding as an indicator for sequence accessibility within the transcripts. Scherr, M., et al., Nucleic Acids Res. 26: 5079-5085,1998; Lloyd, et al., Nucleic Acids Res. 29: 3665-3673, 2001. A mixture of antisense oligonucleotides that are complementary to certain MASP-2 transcribed regions is added to the MASP-2 expressing cell extracts, such as hepatocytes. and hybridized to create a RNAseH vulnerable site. This method may be combined with computer-aided selectivity that may predict optimal selectivity for antisense compositions based on their relative ability to form dimers, hairpins, or other secondary structures that would reduce or prevent specific binding to target mRNA. in a host cell. This secondary structure analysis and target site selection considerations can be performed using the OLIGO primer analysis software (Rychlik, I., 1997) and the BLASTN 2.0.5 algorithm software (Altschul, SF, et al., Nucl. Acids Res 25: 3389-3402,1997). The antisense compounds directed against the sequence preferably comprise from about 8 to about 50 nucleotides at length. Antisense oligonucleotides comprising from about 9 to about 35 or nucleotides are particularly preferred. The inventors consider all oligonucleotide compositions in the range of 9 to 35 nucleotides (i.e. those of approximately 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 bases in length) are highly preferred for practicing antisense oligonucleotide based methods of the invention. The highly preferred target regions of the MASP-2 mRNA are those that are in or near the AUG translation start codon and those sequences that are substantially complementary to the 5 'regions of the mRNA, for example, between the -10 and +10 regions of the nucleotide of the MASP-2 gene (SEQ ID NO: 4). Exemplary MASP-2 expression inhibitors are provided in TABLE 4.

Table 4: Exemplary MASP-2 Expression Inhibitors

<table> table see original document page 143 </column> </row> <table>

As mentioned above, the term "oligonucleotide" as used herein refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term also encompasses those oligonucleobases composed of naturally occurring nucleotide, sugars, and covalent internucleoside bonds (main chain) as well as oligonucleotides having naturally occurring modifications. These modifications allow the introduction of certain desirable properties that are not offered through naturally occurring polyolucleotides, such as reduced toxic properties, increased stability against nuclease degradation, and enhanced cell uptake. In illustrative embodiments, the antisense compounds of the invention differ from native DNA by modifying the phosphodiester backbone to extend the life of the antisense oligonucleotide in which phosphate substituents are replaced by phosphorothioates. Likewise, one or both ends of the oligonucleotide may be replaced by one or more acridine derivatives that intersect adjacent base pairs within a nucleic acid strand.

Another alternative to antisense is the use of "RNA interference" (RNAi). Double stranded RNAs (dsRNAs) can cause gene silencing in mammals in vivo. The natural function of RNAi and co-suppression appear to be protecting the genome against invasion by mobile genetic elements such as retrotransposons and viruses that produce aberrant RNA or dsRNA in the host cell when they become active (see, for example, Jensen, J., et al. Nat. Genet 21: 209-12,1999). The double stranded RNA molecule can be prepared by synthesizing two RNA strands capable of forming a double stranded deRNA molecule, each having a length of about 19 to (e.g., 19 to 23 nucleotides). for example, a dedsRNA molecule useful in the methods of the invention may comprise RNA corresponding to a sequence and its complement listed in TABLE 4.

Preferably, at least one RNA strand has a 3 'projection of 1 to 5 nucleotides. The synthesized RNA strands are combined under conditions that form a double stranded molecule. The deRNA sequence may comprise at least a portion of 8 nucleotides of SEQID NO: 4 with a total length of 25 nucleotides or less. The planning of siRNA sequences for a given target is within the ordinary skill of the person in the art. Commercial services are available that plan the siRNA sequence and guarantee at least 70% disillusionment of the expression (Qiagen, Valencia, Calif).

The dsRNA may be administered as a pharmaceutical composition and performed by known methods, wherein a nucleic acid is introduced into a desired target cell. Commonly used degene transfer methods include calcium phosphate, DEAE-dextran, electroporation, microinjection and viral methods. Such methods are disclosed in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons3 Inc., 1993.

Ribozymes may also be used to decrease the amount and / or biological activity of MASP-2, such as ribozymes that target MASP-2 mRNA. Ribozymes are catalytic RNA molecules that can cleave nucleic acid molecules having a sequence that is completely or partially homologous to the ribozyme sequence. It is possible to design ribozyme transgenes that encode RNA ribozymes that specifically pair with a target RNA and cleave the phosphodiester main chain at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, ribozyme is not altered by itself and is thus able to recycle and cleave other molecules. Inclusion of ribozyme sequences within antisense RNAs gives RNA cleavage reactivity to them, thereby increasing the activity of antisense constructs.

Ribozymes useful in the practice of the invention typically comprise a hybridization region of at least about novenucleotides, which is complementary in nucleotide sequence to at least part of the target MASP-2 mRNA, and a catalytic region that is adapted to cleave MASP-2 mRNA target (see generally EPA No. 321 321 201; W088 / 04300; Haseloff, J., et al., Nature 334: 585-591, 1988; Fedor, M.J., et., Proc. Natl. Acad. Sci. USA 87: 1668-1672, 1990; Cech, TR, et al., Ann.Rev. Biochem. 55: 599-629 (1986).

Ribozymes can be targeted directly to cells in the form of RNA oligonucleotides incorporating ribozyme or sequences introduced into the cell as an expression vector encoding the desired deribozyme RNA. Ribozymes may be used and applied in large part in the same manner as described for antisense polynucleotides.

Antisense RNA and DNA, ribozymes, and RNAutile molecules in the methods of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art, such as for example chemical synthesis of solid phase phosphoramidite. Alternatively, RNA molecules may be generated by in vitro transcription and in vivo DNA sequences that encode the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors incorporating suitable RNA polymerase promoters such as T7 or SP6 polymerase promoters. Alternatively, antisense decDNA constructs that synthesize constitutive antisense RNA or inductively, depending on the promoter used, may be introduced stably into cell lines.

Several well-known modifications of DNA molecules may be introduced as a means of increasing stability and half-life. Useful modifications include, but are not limited to the addition of flanking ribonucleotide or deoxyribonucleotide sequences to the 5 'and / or 3' termini of the molecule or the use of phosphorothioate or 2'-0-methylation rather than phosphodiesterase linkages within the deoligodeoxyribonucleotide backbone.

V. PHARMACEUTICAL COMPOSITIONS AND DOSAGE RELEASE METHODS

In another aspect, the invention provides compositions for inhibiting the adverse effects of MASP-2-dependent complement activation comprising a therapeutically effective amount of a MASP-2 inhibitor and a pharmaceutically acceptable carrier. MASP-2 inhibiting agents may be administered to a patient in need thereof at therapeutically effective doses to treat or ameliorate conditions associated with MASP-2 dependent complement activation. A therapeutically effective dose refers to the amount of inhibitor DEMASP-2 sufficient to result in amelioration of symptoms of the condition.

The toxicity and therapeutic efficacy of the DEMASP-2 inhibitory agents can be determined by standard pharmaceutical procedures using experimental animal models, such as the MASP-2 - / - mouse mouse model expressing the DEMASP-2 human transgene described in Example 3. Using Such animal models, NOAEL (no observed adverse effect level) and MED (the minimally effective dose) can be determined using standard methods. The dose ratio between NOAEL and MED effects is the therapeutic ratio, which is expressed as the NOAEL / MED ratio. MASP-2 inhibiting agents exhibiting ratios or large therapeutic indices are more preferred. Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. Dosing of the MASP-2 inhibitory agent preferably resides within a range of circulating concentrations that include MED with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used.

For any compound formulation, the therapeutically effective dose can be estimated using animal models. For example, a dose may be formulated in an animal model to achieve a circulating plasma concentration range that includes MED. Quantitative levels of MASP-2 inhibitor in plasma can also be measured, for example, by high performance liquid chromatography.

In addition to toxicity studies, the effective dosage can also be estimated based on the amount of MASP-2 protein present in a live patient and the binding affinity of the MASP-2 inhibiting agent. It has been shown that MASP-2 levels in patients Normal humans are present in serum at low levels in the range of 500 ng / ml and the levels of MASP-2 in a particular patient can be determined using a quantitative MASP-2 assay described in Moller-Kristensen M., et al., J. Immunol. Methods 282: 159-167, 2003.

In general, the dosage of compositions administered comprising MASP-2 inhibiting agents varies depending on factors such as age, weight, height, gender, general medical condition and prior medical history of the patient. As an illustration, MASP-2 inhibitory agents, such as anti-MASP-2 antibodies, may be administered in tapering ranges of about 0.010 to 10.0 mg / kg, preferably from 0.010 to 1.0mg / kg, more preferably. 0.010 to 0.1 mg / kg of the patient's body weight. In some embodiments the composition comprises a combination of anti-MASP-2 antibodies and MASP-2 inhibiting peptides.

The therapeutic efficacy of MASP-2 inhibitor compositions and methods of the present invention in a given patient and the appropriate dosages may be determined according to complement assays well known to those of skill in the art. The complement generates many specific products. Over the last decade, sensitive and specific assays have been developed and commercially available for most of these activation products, including fragments of. small C3a, C4a and C5a activation and the large activation fragments iC3b, C4d, Bbe sC5b-9. Most of these assays use monoclonal antibodies to target new antigens (neoantigens) exposed on the fragment, but not the native proteins from which they are formed, making these assays very simple and specific. Most rely on ELISA technology, although radioimmunoassay is still sometimes used for C3ae C5a. These latter tests measure both unprocessed fragments and their 'desArg' fragments, which are the main forms found in the circulation. Unprocessed C5adeSArg fragments are rapidly cleared by binding to cell surface receptors and are present at very low concentrations, whereas C3adeSArg does not cell-bind and accumulate in plasma. C3a measurements provide a sensitive indicator regardless of the path of complement activation. Alternate path activation can be assessed by measuring fragmentBb. Detection of the fluid phase product of sC5b-9 activation of the membrane attack pathway provides evidence that the complement is being activated until completion. Because both the lectin pathway and the classical pathway generate the same activation products, C4a and C4d, measurements of these two fragments did not provide any information about which of these two pathways generated the activation products.

ADDITIONAL AGENTS

Compositions and methods comprising MASP-2 inhibitory agents may optionally comprise one or more additional therapeutic agents, which may enhance the activity of the MASP-2 inhibitory agent or which provide related therapeutic functions in an additive or synergistic manner. For example, one or more MASP-2 inhibiting agents may be administered in combination with one or more anti-inflammatory and / or analgesic agents. The inclusion and selection of additional agent (s) will be determined to obtain a desired therapeutic result. Suitable antiinflammatory and / or analgesic agents include: serotonin receptor antagonists; serotonin doreceptor agonists; histamine receptor antagonists; bradykinin doreceptor antagonists; kallikrein inhibitors; detaquicinin receptor antagonists, including neurokinin and eneurokinin2 receptor subtype antagonists; dacalcitonin gene-related peptide receptor (CGRP) antagonists; interleukin receptor antagonists; synthetic pathway-active enzyme inhibitors for arachidonic acid metabolites, including phospholipase inhibitors, including PLA2 isoform inhibitors and PLC7 isoform inhibitors, cyclooxygenase (COX) inhibitors (which may be COX-1, COX-2, or non-selective COX inhibitors) -Ie -2), lipooxygenase inhibitors; prostanoid receptor antagonists including EP-I and EP-4 receptor subtype antagonists and thromboxane receptor subtype eicosanoid antagonists; leukotriene receptor antagonists includinglaukotriene B4 receptor subtype antagonists and leukotriene D4 receptor subtype antagonists; opioid receptor agonists, including agonists of the opioid receptor subtype, opioid δ and opioid κ; purinoceptor agonists and antagonists including P2x and P2y receptor antagonists; adenosine triphosphate (ATP) sensitive potassium channel openers; MAP kinase inhibitor, nicotinic acetylcholine inhibitors; and alpha adrenergic receptor agonists (including alpha-1, alpha-2 and non-selective alpha-1 and 2 agonists).

When used in the prevention or treatment of restenosis, the MASP-2 inhibitor agent of the present invention may be combined with one or more anti-restenosis agents for concomitant administration. Suitable anti-restenosis agents include: antiplatelet agents including: thrombin inhibitors and receptor antagonists, adenosine dodiphosphate receptor (ADP) antagonists (also known as purinoceptor 1 doreceptor antagonists), thromboxane inhibitors and dereceptor antagonists, and receptor antagonists. platelet membrane glycoprotein; cell adhesion molecule inhibitors, including deselectin inhibitors and integrin inhibitors; anti-chemotactic agents; interleukin doreceptor antagonists; and cell signaling inhibitors including: protein kinase C (PKC) inhibitors and protein tyrosine phosphatases, modulators of intracellular protein tyrosine kinase inhibitors, inhibitors of SRC (SH2) domains and calcium channel antagonists.

The MASP-2 inhibiting agents of the present invention may also be administered in combination with one or more other complement inhibitors. None of the complement inhibitors is currently approved for use in humans, however some pharmacological agents have been shown to block complement in vivo. Many of these agents are also toxic or are only partial inhibitors (Asghar, S. S., Pharmacol. Rev. 36: 223-44, 1984) and the dose of use has been limited to resting as research tools. K76COOH and Nafamstat Mesylate are two agents that have shown some efficacy in animal transplant models (Miyagawa, S., et al., Transplant Proc. 24: 483-484, 1992). Low molecular weight heparins have also been shown to be effective in regulating complement activity (Edens, R.E., et al., ComplementToday, pp. 96-120, Basel: Karger, 1993). These small demolecule inhibitors are believed to be useful as agents for use in combination with the MASP-2 inhibiting agents of the present invention.

Other naturally occurring complement inhibitors may be useful in combination with the MASP-2 inhibiting agents of the present invention. Biological complement inhibitors include soluble complement factor 1 (sCRl). This is a naturally occurring inhibitor that can be found in the outer membrane of human cells. Other membrane inhibitors include DAF5 MCP and CD59. Recombinant formations were tested for their anti-complement activity in vitro and in vivo. sCRl has been shown to be effective in transplantation, in which the complement system (both quantoclassical alternative) provides the trigger for a hyperactive rejection syndrome within minutes of perfused blood through the newly transplanted organ (Platt JL, et al, Immunol. Today / /: 450-6, 1990; Marino IR, et al., Transplant Proc. 1071: 6 (1990; Johnstone, PS, et al., Transplantation 54: 573-6, 1992). The use of sCRl protects and prolongs the transplanted organ's survival time, implying the complement pathway in the pathogenesis of organ survival (Leventhal, JR et al., Transplantation 55: 857-66,1993; Pruitt, SK, et al., Transplantation). 57: 363-70, 1994).

Additional complement inhibitors suitable for use in combination with the compositions of the present invention also include, for example, MoAbs such as those developed by Alexion Pharmaceuticals, Inc., New Haven, Connecticute anti-properdin MoAbs.

When used in the treatment of arthritis (e.g., osteoarthritis and rheumatoid arthritis), the MASP-2 inhibiting agent of the present invention may be combined with one or more chondroprotective agents, which may include one or more cartilage anabolism promoters and / or one or more cartilage catabolism inhibitors and suitably both an anabolic agent and a catabolic inhibitory agent for concomitant administration. Suitable anabolic chondroprotective promoting agents include interleukin (IL) receptor agonists including IL-4, IL-10, IL-13, rhIL-4, rhIL-10 and rhIL-13 and IL-4, IL-10 or IL-13 chimeric; transforming growth factor superfamily agonists including TGF-β, TGF-βΙ, TGF-P2, TGF-β3, bone morphogenic proteins including BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 ( OP-I) and OP-2 / BMP-8, growth-differentiating factors including GDF-5, GDF-6 and GDF-7, TGF-Ps recombinant eBMPs and TGF-ps and chimeric BMPs; insulin-equivalent growth factors including IGF-1; and fibroblast growth factors including bFGF. Suitable inhibitory chondroprotective agents include Interleukin-1 (IL-1) receptor (IL-IRA) antagonists, including human soluble IL-I (shuIL-1R) receptors, rshuIL-1R, rhlL-1ra, anti-IL1 antibody. , AFl 1567 and AF12198; Tumor Necrosis Factor Receptor (TNF) (TNF-α) Antagonists, including soluble receptors including sTNFRI and sTNFRII, soluble TNFrecombinant receptors and soluble chimeric TNF receptors: chimeric Fc, soluble fusion Fc receptors and TNF: cyclooxygenase-2 (COX-2 specific) inhibitors including DuP 697, SC-58451, celecoxib, rofecoxib, nimesulide, diclofenac, meloxicam, piroxicam, NS-398, RS-57067, SC-57666, SC-58125 , flosulide, etodolac, L-745,337 and DFU-T-614; Mitogen-activated protein kinase (MAPK) inhibitors including ERK1, ERK2, SAPK1, SAPK2a, SAPK2b, SAPK2d, SAPK3 inhibitors including SB 203580, SB 203580 iodine, SB202190, SB242235, SB 220025, RWJ 67657, RWJ 68354, FR 133605, L-167307, PD98059, PD 169316; kappa nuclear factor (NFkB) inhibitors, including caffeic acid phenylethyl ether (CAPE), DM-CΑΡΕ, SN-50 peptide, hymenialdysine and pyrrolidone dithiocarbamate; nitric oxide synthase (NOS) inhibitors including NG-monomethyl-L-arginine, 1400W, diphenyleneiodide, S-methyl isothiourea, S- (aminoethyl) isothiourea, L-N6- (I-Iminoethyl) Iysine, 1,3-PBITU, 2-ethyl-2-thiopseudourea, aminoguanidine, Νω-ηίΐΓ0-L-arginine methyl ester and N®-nitro-L-arginine, matrix metalloproteinases (MMPs) inhibitors, including MMP-1, MMP-2 inhibitors, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14 and MMP-15 including U-24522, Minocycline, 4-Abz- Gly-Pro-D-Leu-D-Ala-NHOH, Ac-Arg-Cys-Gly-Val-Pro-Asp-NH 2, rhuman TIMP1, rhuman TIMP2 and phosphoramidon; cell adhesion molecules, including integrin agonists and antagonists including ανβ3 MoAb LM 609 and echistatin; anti-chemotactic agents including F-Met-Leu-Phe receptors, IL-8 receptors, MCP-I receptors, and MIP1-I / RANTES receptors; intracellular signaling inhibitors, including (a) protein kinase inhibitors, including both (i) protein kinase C (PKC) (isozyme) inhibitors includingocalphostin C, G-6203 and GF 109203X and (ii) protein tyrosine kinase inhibitors; (b) intracellular protein tyrosine phosphatase modulators (PTPases); and (c) SH2 domain inhibitors (Homology2 src domains).

For some applications, it may be beneficial to administer the MASP-2 inhibitor agents of the present invention in combination with a spasm inhibitor agent. For example, for urogenital applications, it may be beneficial to include at least one muscle-spasm spasm inhibiting agent and / or at least one anti-inflammatory agent and for vascular procedures it may be useful to include at least one vasospasm inhibitor and / or at least one anti-inflammatory agent. -inflammatory and / or at least one anti-restenosis agent. Suitable examples of spasm inhibiting agents include: serotonin2 receptor subtype antagonists; tachykinin receptor antagonists; donors of nitric oxide; ATP-sensitive potassium channel openers; calcium channel antagonists; and endothelin receptor antagonists.

PHARMACEUTICAL RELEASE VEHICLES AND VEHICLES

In general, the MASP-2 inhibiting agent compositions of the present invention, combined with any other selected therapeutic agents, are suitably contained in a pharmaceutically acceptable carrier. The vehicle is non-toxic, biocompatible and is selected so as not to adversely affect the biological activity of the MASP-2 inhibiting agent (and any other therapeutic agents combined with it). Exemplary pharmaceutically acceptable carriers for the peptides are described in U.S. Patent No. 5,211,657 issued to Yamada. Antibodies-MASP-2 and inhibitor peptides useful in the invention may be formulated into solid, semi-solid, gel, liquid or gaseous preparations such as tablets, capsules, powders, granules, ointments, solutions, deposits, inhalants and injections which allow oral administration, parenteral or surgical. The invention also contemplates local administration of the compositions covering medical and other devices.

Suitable vehicles for parenteral release via injection, infusion or topical release include distilled water, physiological phosphate buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution or propanediol. In addition, sterile, fixed oils may be used as a solvent or suspending medium. For this purpose any oleobiocompatible oil may be used including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be composed as a liquid, suspension, polymerizable or non-polymerizable gel, paste or ointment.

The vehicle may also comprise a release vehicle for sustaining (i.e. prolonging, delaying or regulating) the release of the agent (s) or for enhancing the release, uptake, stability or pharmacokinetics of the therapeutic agent (s) ( s). Such a delivery vehicle may include, by way of non-limiting example, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles. Suitable hydrogel and micelle deliberation systems include the PEO: PHB: PEO copolymers and copolymer / cyclodextrin complexes disclosed in WO2004 / 009664 A2 and the PEO and PEO / cyclodextrin complexes disclosed in US 2002/0019369 A1. Such hydrogels may be injected locally into intended site of action either subcutaneously or intramuscularly to form a sustained release depot.

For intraarticular release, the MASP-2 inhibitory agent may be charged in the above described liquid or gel vehicles which are injectable, sustained release vehicles described above which are injectable or a hyaluronic acid or hyaluronic acid derivative.

For oral administration of non-peptideric agents, the MASP-2 inhibitor may be loaded into an inert filler or diluent such as sucrose, cornstarch or cellulose.

For topical administration, the MASP-2 inhibitory agent may be loaded into ointment, lotion, cream, gel, gout, suppository, spray, liquid or powder or gel or microcapsular delivery systems via a transdermal patch.

Various nasal and pulmonary delivery systems, including aerosols, metered dose inhalers, dry powder inhalers and nebulizers, are being developed and may be suitably adapted for release of the present invention into an aerosol, inhalant or nebulized delivery vehicle, respectively.

For intrathecal (IT) or intracerebroventricular (ICV) release, appropriately sterile delivery systems (e.g., liquids; gels, suspensions, etc.) may be used to administer the present invention.

Compositions of the present invention may also include biocompatible excipients such as dispersing or wetting agents, suspending agents, diluents, buffers, penetration enhancers, emulsifiers, binders, thickeners, flavoring agents (for oral administration).

PHARMACEUTICAL VEHICLES FOR ANTIBODIES AND PEPTIDES

More specifically with respect to anti-MASP-2 antibodies and inhibitor peptides, exemplary formulations may generally be administered as injectable dosages of a solution or suspension of the compound in a physiologically acceptable diluent with a pharmaceutical carrier which may be a sterile liquid such as water, oils, saline, glycerol or ethanol. Additionally, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and others may be present in the compositions comprising anti-MASP-2 antibodies and inhibitor peptides. Additional components of pharmaceutical compositions include petroleum (such as animal, vegetable or synthetic origin), for example soybean oil and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers for injectable solutions.

Anti-MASP-2 antibodies and inhibitor peptides may also be administered in the form of an injection preparation or depot implant which may be formulated in such a manner as to permit sustained or pulsatile release of the active agents.

PHARMACEUTICALLY ACCEPTABLE VEHICLES FOR EXPRESSION INHIBITORS

More specifically with respect to expression inhibitors useful in the methods of the invention, compositions are provided which comprise an expression inhibitor as described above and a pharmaceutically acceptable carrier or diluent. The composition may further comprise a colloidal dispersion system.

Pharmaceutical compositions which include expression inhibitors may include, but are not limited to, liposome-containing solutions, emulsions and formulations. These compositions may be generated from a variety of components including, but not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semi-solids. Preparation of such compositions typically involves combining the expression inhibitor with one or more of the following: buffers, antioxidants, low molecular weight polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are examples of suitable diluents.

In some embodiments, the compositions may be prepared and formulated as emulsions which are typically heterogeneous systems of one liquid dispersed in another in the form of droplets (see, Idson, Pharmaceutical Dosage Forms, Vol. 1, Rieger and Banker (eds.)). , Marcek Dekker, Inc., NY, 1988). Examples of naturally occurring emulsifiers used in emulsion formulations include acacia, beeswax, lanolin, lecithin and phosphatides.

In one embodiment, compositions including nucleic acids may be formulated as microemulsions. A microemulsion as used herein refers to a water, oil and amphiphile system which is an optically isotropic and thermodynamically stable liquid solution (see Rosoff in Pharmaceutical Dosage Forms, Vol. 1). The method of the invention may also use liposomes for the transfer and release of antisense oligonucleotides to the desired site.

Expression inhibitor pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, lozenges, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, as well as aqueous, powdered or oily bases and thickeners and others may be used.

Pharmaceutical compositions comprising MASP-2 inhibiting agents may be administered in various modes depending on whether a local or systemic mode of administration is more appropriate for the condition being treated. Additionally, as described hereinabove with respect to extracorporeal reperfusion procedures, MASP-2 inhibiting agents may be administered by introducing the compositions of the present invention into recirculating blood or plasma. In addition, the compositions of the present invention may be released by coating or incorporating the compositions on or within an implantable medical device.

SYSTEMIC RELEASE

As used herein, the terms "systemic release" and "systemic administration" are intended to include but are not limited to oral and parenteral routes including intramuscular (IM), subcutaneous, intravenous (IV), intraarterial, inhalation, sublingual, buccal, Topical, transdermal, nasal, rectal, vaginal and other routes of administration that effectively result in the release of the released agent to a single or multiple site of intended therapeutic action. Preferred systemic release routes for the present compositions include intravenous, intramuscular, subcutaneous and inhalational. It will be appreciated that the exact systemic route of administration for the selected agents used in particular compositions of the present invention will be determined in part by taking into account the susceptibility of the agent to metabolic transformation pathways associated with a given route of administration. For example, peptide agents may be more suitably administered by routes other than oral.

MASP-2 inhibitor antibodies and polypeptides may be released to a patient in need thereof by any suitable means. MASP-2 antibody and polypeptide release methods include oral, pulmonary, parenteral (eg, intramuscular, intraperitoneal, intravenous (IV) or subcutaneous) administration, inhalation (such as through a fine powder formulation). ), transdermal, nasal, vaginal, rectal or sublingual administration and may be formulated in dosage forms appropriate for each administration route.

By way of representative example, MASP-2 inhibiting antibodies and peptides may be introduced into a living body by application to a body membrane capable of absorbing polypeptides, such as nasal, gastrointestinal and rectal membranes. Polypeptides are typically applied to the absorptive membrane in conjunction with a permeation enhancer. (See, for example, Lee, VHL, Crit. Rev. Ther. Drug CarrierSys. 5: 69, 1988; Lee, VHL, J. Controlled Release 13: 213, 1990; Lee, VHL, Ed., Peptide and Protein Drug Delivery, Mareei Dekker, New York (1991); DeBoer, AG, et al., J. Controlled Release, 13: 241, 1990). For example, STDHF is a synthetic derivative of fusidic acid, a steroidal surfactant that is similar in structure to bile salts and has been used as a permeation enhancer for nasal release. (Lee, W.A., Biopharm.22, Nov./Dec. 1990).

MASP-2 inhibitor antibodies and polypeptides may be introduced in combination with another molecule, such as a lipid, to protect the polypeptides from enzymatic degradation. For example, covalent polymeric targeting, especially polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body and thus prolong half-life (Fuertges, F., et al., J. Controlled Release 11: 139, 1990). ). Many polymeric systems have been reported for protein release (Bae, YH, et al., J. Controlled Release 9: 271, 1989; Hori, R., etal., Pharm. Res. 6: 813, 1989; Yamakawa, I , J. et al., J. Pharm. Sci. 79: 505,1990; Yoshihiro, I., et al., J. Controlled Release 10: 195, 1989; Asano, M., etal., J. Controlled Release 9 : 111, 1989; Rosenblatt, J., et al., J. Controlled Release 9: 195, 1989; Makino, K., J. Controlled Release 12: 235, 1990; Takakura, Y., et al., J. Pharm Sci. 78: 117, 1989; Takakura, Y., et al., J. Sharm. Sci. 78: 219, 1989).

Recently, liposomes have been developed with improved serum stability and circulating half-lives (see, for example, U.S. Patent No. 5,741,516 issued to Webb). In addition, various methods of liposome and liposome equivalent preparations with potential drug vehicles have been reviewed (see, for example, US Patent No. 5,567,434 issued to Szoka; US Patent No. 5,552,157 issued to Yagi; US Patent No. 5,565,213 issued to Nakamori; U.S. Patent No. 5,738,868 issued to Shinkarenko; and US Patent No. 5,795,587 to Gao).

For transdermal applications, MASP-2 inhibitor antibodies and polypeptides may be combined with other suitable ingredients, such as vehicles and / or adjuvants. There is no limitation on the nature of such other ingredients except that they should be pharmaceutically acceptable as to their intended administration and may not degrade the activity of the active ingredients of the composition. Examples of suitable carriers include ointments, creams, gels or suspensions, with or without purified collagen. MASP-2 inhibitor antibodies and polypeptides may also be impregnated in plasmatic transdermal patches, plasters and bandages, preferably in liquid or semi-liquid forms.

The compositions of the present invention may be systematically administered on a periodic basis at certain intervals to maintain a desired level of therapeutic effect. For example, compositions may be administered, such as by subcutaneous injection, every two to four weeks or at less frequent intervals. The dosage regimen will be determined by the physician considering various factors that may influence the action of the combination of agents. These factors will include the degree of progress of the condition being treated, the age, gender and weight of the patient, and other clinical factors. The dosage for each individual agent will vary as a function of the MASP-2 inhibiting agent that is included in the composition, as well as the presence and nature of any drug deliberation vehicle (for example, a sustained release vehicle). Dosage may be adjusted taking into account the variation in the frequency of administration and the pharmacokinetic behavior of the released agent (s).

LOCAL RELEASE

As used herein, the term "site" encompasses the application of a drug to or around a localized site of action and may include, for example, topical release to the skin or other affected tissues, ophthalmic release, administration, placement or intrathecal irrigation (IT). ), intracerebroventricular (ICV), intraarticular, intracavity, intracranial or intravesicular. Local administration may be preferred to allow administration of a lower dose, to avoid systemic side effects and to more precisely control the release time and concentration of active agents at the local release site. Local administration provides a known concentration at the target site, regardless of interpatient viability in metabolism, blood flow, etc. Enhanced dosage control is also provided by the direct release mode.

Local release of a MASP-2 inhibitory agent may be achieved in the context of surgical methods for treating a disease or condition, such as during procedures such as arterial bypass surgery, atherectomy, laser procedures, ultrasound procedures, angioplasty of balloon and probe placement. For example, a MASP-2 inhibitor may be administered to a patient in conjunction with a balloon angioplasty procedure. A weak angioplasty procedure involves inserting a catheter having a deflated balloon inside an artery. The deflated balloon is positioned near the atherosclerotic plaque and is inflated such that the plaque is pressed against the wall of the vein. As a result, the balloon surface is in contact with the bed of vascular endothelial cells on the blood vessel surface. The MASP-2 inhibitor agent may be attached to the balloon angioplasty catheter in a manner that allows release of the agent at the atherosclerotic plaque site. The agent may be attached to the balloon catheter according to standard procedures known in the art. For example, the agent may be stored in a balloon catheter compartment until the balloon is deflated, at which point it is released into the local environment. Alternatively, the agent may be impregnated on the surface of the balloon such that it contacts the arterial wall cells as the balloon is inflated. The agent may also be delivered into a perforated balloon catheter such as those disclosed in Flugelman, MY, et al., Circulation 85: 1110-1117, 1992. Also published PCT Application WO 95/23161 for an exemplary procedure for binding a therapeutic protein to a weak angioplasty catheter. Likewise, the MASP-2 inhibiting agent may be included in a gel or polymeric coating applied to a probe or may be incorporated into the probe material such that the probe elutes the DEMASP-2 inhibiting agent after vascular placement.

MASP-2 inhibitor compositions used in the treatment of arthritis and other musculoskeletal disorders may be locally released by intraarticular injection. Such compositions may suitably include a sustained release vehicle. As another example of cases where local release may be desired, MASP-2 inhibitor compositions used in the treatment of urogenital conditions may be suitably instilled intravesically or within another urogenital structure.

COATINGS IN A MEDICAL DEVICE

MASP-2 inhibitory agents such as inhibitory epeptide antibodies may be immobilized on (or within) a surface of an implantable or pluggable medical device. The modified surface will typically be in contact with living tissue after implantation into an animal body. By "implantable or pluggable medical device" is meant any device that is implanted in or attached to the tissue of an animal body during normal operation of the device (e.g., probes and implantable drug delivery devices). Such implantable or pluggable medical devices may be manufactured, for example, from nitrocellulose, diazocellulose, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, dextran, sepharose, agar, starch, nylon, stainless steel, titanium and biodegradable and / or biocompatible polymers. Binding of the protein to a device may be performed by any technique that does not destroy the biological activity of the bound protein, for example by ligating one or both of the N or C-terminal residues of the protein to be delivered. Binding may also be done at one or more internal sites on the protein. Multiple bonds (both internal and at the ends of the protein) may also be used. A surface of an implantable or pluggable medical device may be modified to include functional groups (e.g., carboxyl, amide, amino, ether, hydroxyl, cyano, nitride, sulfanamide, acetylinic, epoxide, silane, anhydrous, succinimic, azide) for protein to this one. Binding chemicals include, but are not limited to, the formation of ester derivatives, ethers, amides, azido and sulfanamide, cyanate and other functional group bonds available on MASP-2 antibodies or inhibitor peptides. MASP-2 antibodies or inhibitor fragments can also be non-covalently linked by the addition of a protein affinity label sequence, such as GST (DB Smith and KS Johnson, Gene 67: 31, 1988), polystidines (E. Hochuli et al. , J. Chromatog. 411: 77, 1987) or biotin. Such affinity labels may be used for reversible protein binding to a device.

Proteins may also be covalently attached to the surface of a device body, for example by covalently activating the surface of the medical device. By way of representative example, the matrix cell protein (s) may be attached to the device body by any of the following pairs of reactive groups (a member of the parasite present on the surface of the device body). device and the other doping member being present in the matricellular protein (s): hydroxyl / carboxylic acid to produce an ester bond; hydroxyl / anhydride to produce an ester bond; hydroxyl / isocyanate to produce a urethane bond. A surface of a device body that lacks useful reactive groups may be treated with radio frequency-discharge plasma (RFGD) recording to generate reactive groups to allow for the adhesion of matricellular protein (s) (e.g., treatment oxygen therapy to introduce oxygen containing groups; propyl amino treatment to introduce amino groups).

MASP-2 inhibitory agents comprising nucleic acid molecules such as peptide inhibitors encoding RNAi or antisense DNA may be embedded in porous arrays attached to a device body. Representative porous matrices useful for fabricating the surface layer are those prepared from tendon or dermal collagen, as can be obtained from a variety of commercial sources (e.g., Sigma and Collagen Corporation) or collagen matrices prepared as described in US Pat. 4,394,370 to Jefferies and 4,975,527 to Koezuka. A decollagen material is called UltraFiber® and is obtainable from Norian Corp. (MountainView, California).

Certain polymeric matrices may also be used if desired and include acrylic ester polymers and lactic acid polymers, as disclosed, for example, in U.S. Patent Nos. 4,526,909 issued to Urist and 4,563,489 to Urist. Particular examples of useful polymers are those of orthoesters, anhydrides, propylene cofiimarates or a polymer of one or more α-hydroxy carboxylic acid monomers (eg α-hydroxy acetic acid (glycolic acid) and / or α-hydroxypropionic acid (acid lactic)).

TREATMENT SCHEMES

In prophylactic applications, the pharmaceutical compositions are administered to a patient susceptible to or otherwise at risk of a condition associated with MASP-2-dependent complement activation in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic applications, the pharmaceutical compositions are administered to a patient suspected of or already suffering from a condition associated with MASP-2 dependent complement activation in a therapeutically effective amount sufficient to alleviate or at least partially reduce the symptoms of the condition. In prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitor agents may be administered in various dosages until sufficient therapeutic result has been obtained in the patient. Application of the MASP-2 inhibitor compositions of the present invention may be performed by a single administration of the composition or a limited sequence of administrations for the treatment of an acute condition, e.g. reperfusion injury or other traumatic injury. Alternatively, the composition may be administered at periodic intervals over an extended period for the treatment of chronic conditions, for example, arthritis or psoriasis. The methods and compositions of the present invention may be used to inhibit inflammation and related processes that typically result from diagnosis and procedures. therapeutic and surgical practitioners. To inhibit such processes, the MASP-2 inhibitor composition of the present invention may be applied peri-procedurally. As used herein "periprocedurally" refers to the administration of the preprocedural and / or intraprocedural and / or postprocedural inhibitory composition, that is, before the procedure, before and during the procedure, before and after the procedure, before, during and after the procedure. procedure, during the procedure, during and after the procedure or after the procedure. Peri-procedural application may be performed by local administration to the surgical or procedural site, such as by continuous or intermittent injection or irrigation of the site or by systemic administration. local perioperative release of MASP-2 inhibitor solutions is disclosed in US Pat. Nos. 6,420,432 issued to Demopulos and 6,645,168 issued to Demopulos. Suitable methods for local release of chondroprotective compositions including MASP-2 inhibitory agent (s) are disclosed in PCT International Patent Application WO 01/07067 A2. Suitable methods and compositions for the targeted systemic release of chondroprotective compositions including MASP-2 inhibitory agent (s) are disclosed in PCT International Patent Application WO 03/063799 A2.

SAW. EXAMPLES

The following examples merely illustrate the best mode now considered for the practice of the invention, but should not be construed as limiting the invention. All literature citations herein are expressly incorporated by reference.

EXAMPLE 1

This example describes the generation of a MASP-2 deficient (MASP-2 - / -) but sufficient strain of MAp19 (MAp19 + / +).

Materials and Methods: The targeting vector pKO-NTKV1901 was designed to disrupt the three exons encoding the murine MASP-2 end-C, including the exon encoding the serine protease domain, as shown in FIGURE 4. PKO-NTKV 1901 was used to transfect murine ES14.1a cell line (SV129Ola). Neomycin resistant and thymidine kinase sensitive clones were selected. 600 ES clones were screened and of these four different clones were identified and verified by the southern blot to contain the expected selective recombination and blending event as shown in FIGURE 4. Chimeras were generated from these four embryo transfer positive clones. The chimeras were then backcrossed into the C57 / BL6 fundogenetic to create transgenic males. Transgenic males were crossed with females to generate Fls with 50% of progeny showing heterozygosity for the disrupted MASP-2 gene. Heterozygous mice were intercrossed to generate insufficient homozygous progeny in MASP-2, resulting in heterozygous and wild type mice in the ratio of 1: 2: 1, respectively.

Result and Phenotype: The resulting MASP-2 - / - deficient homozygous mice were found to be viable and fertile and found to be MASP-2 deficient by the southern blot to confirm the correct targeting event by the Northern blot to confirm the absence of MASP-2 mRNA. 2 and Western blot to confirm the absence of MASP-2 protein (data not shown). The presence of MAp19 demRNA and the absence of MASP-2 mRNA were further confirmed using time-resolved RT-PCR on a LightCycler machine. MASP-2 - / - mice continued to express MAp19, MASP-1 and MASP-3 mRNA and protein as expected (data not shown). The presence and abundance of mRNA in MASP-2 - / - mice for Properdin, Factor B, Factor D, C4, C2, and C3 were assessed by LeveCycler analysis and found to be identical to those of wild type denoted controls (data not shown). . MASP-2 - / - mouse plasma is totally deficient in lectin pathway-complemented complement activation and alternative pathway complement activation as further described in Example 2.

Generating a MASP-2 - / - strain on a pure C57BL6 background: MASP-2 - / - mice are backcrossed with a pure C57BL6 strain for nine generations before using the MASP-2 - / - strain as an animal model. experimental.

EXAMPLE 2

This example demonstrates that MASP-2 is required for complement activation via the alternate and dalectin pathways.

Methods and Materials:

Lectin Path Specific C4 Cleavage Assay: A C4 cleavage assay has been described by Petersen, et al., J. Immunol.Methods 257: 107 (2001) who measured activation of the lipoteichoic acid (LTA) lectin pathway S. aureus, which binds L-ficoline. The assay described in Example 11 was adapted to measure dalectin pathway activation via MBL by plating the plate with LPS and ouzimosan mannan prior to the addition of MASP-2 - / - mouse serum as described below. The assay was also modified to remove the possibility C4 slope due to the classical path. This was achieved using a sample dilution buffer containing 1 M NaCl, which allows high affinity binding of lectin pathway recognition components to their ligands but prevents endogenous C4 activation, thus excluding participation of the classical pathway by complex dissociation. Briefly described, in the modified assay serum samples (diluted in high salt buffer (1 M NaCl)) are added to ligand-coated plates, followed by the addition of a constant amount of purified C4 in a buffer with a physiological salt concentration. . Bound recognition complexes containing MASP-2 cleave C4, resulting in C4b deposition.

Test Methods:

1) Nunc Maxisorb microtiter plates (Maxisorb, Nunc5Cat. No. 442404, Fisher Scientific) were coated with 1 μg / ml mannan (M7504 Sigma) or any other ligand (for example, as listed below) diluted in coating buffer ( 15 mM Na 2 COs, 35 mM NaHCO 3, pH 9.6).

The following reagents were used in the assay:

The. mannan (1 μ§ΔΌ5εηΉΐόπο of mannan (M7504 Sigma) in 100 μΐ of coating buffer);

B. zimosan (1 μg / zimosan reservoir (Sigma) in 100 μΐ of coating buffer);

ç. LTA (1 μg / reservoir in 100 μΐ coating buffer or 2 μg / reservoir in 20 μΐ methanol)

d. 1 μg of specific Mab 4H5 in H-pholine in coating buffer

and. Aerococcus viridans PSA (2 μg / well in 100 μΐ coating buffer)

f. 100 μΐ / S. aureus DSM20233 reservoir fixed in formalin (OD550 = 0.5) in coating buffer.

2) The plates were incubated overnight at 4 ° C.

3) After overnight incubation, residual protein binding sites were saturated by incubating the plates with 0.1% HSA-TBS blocking buffer (0.1% (w / v) HSA in 10 mM Tris-Cl , 140 mM NaCl, 1.5 mM NaN 3, pH 7.4) for 1 to 3 hours, then washing the 3X plates with TBS / tween / Ca2 + (TBS with 0.05% Tween 20 and 5 mM CaCl2, 1 mM MgCl 2, pH 7.4).

4) Serum samples to be tested were diluted in MBL binding buffer (1 M NaCl) and diluted samples were added to the plates and incubated overnight at 4 ° C. Reservoirs receiving buffer were only used as negative controls.

5) Following overnight incubation at 4 ° C, the plates were washed 3X with TBS / tween / Ca2 +. Human C4 (100 μΐ / 1 μΐ diluted BBS reservoir (4 mM barbital, 145 mM NaCl5 2 mM CaCl2, 1 mM MgCl2, pH 7.4)) was then added to the plates and incubated for 90 minutes at 37 ° C . The plates were washed once again 3X with TB S / tween / Ca2 +.

6) C4b deposition was detected with an alkaline phosphatase conjugated anti-human chicken C4c (diluted 1: 1000 in TBS / tween / Ca), which was added to the plates and incubated for 90 minutes at room temperature. The plates were then washed once again 3X withTBS / tween / Ca2 +.

7) Alkaline phosphatase was detected by the addition of 100 μΐ of p-nitrophenyl phosphate substrate, incubating at room temperature for 20 minutes and reading the OD40S on a multi-plate plate reader.

Results: FIGURES 6A-B show the amount of C4b deposition in mannan (FIGURE 6A) and zymosan (FIGURE 6B) in MASP-2 + / + (crosses), MASP-2 +/- (closed circles) and MASP- 2 - / - (closed triangles). FIGURE 6C shows relative C4convertase activity in zymosan (white bars) or mannan (shaded bars) coated plates of MASP-2 - / + (n = 5) mice and MASP-2 - / - (n = 4) mice wild type mice (n = 5) based on measurement of the amount of C4b deposition normalized to wild type serum. Error bars represent standard deviation. As shown in FIGURES 6A-C, MASP-2 - / - mouse plasma is totally deficient in dalectin pathway complement activation in mannan and zymosan coated plates. These results clearly demonstrated that MASP-2, but not MASP-I or MASP-3, is the effector component of the lectin pathway.

C3b Deposition Test:

1) Nunc Maxisorb microtiter plates (Maxisorb, Nunc, cat. No. 442404, Fisher Scientific) are coated with 1μg / mannan reservoir (M7504 Sigma) or any other diluted ligand in coating buffer (15 mM Na2CO3, 35 mM NaHCO 3, pH 9.6) and incubated overnight at 4 ° C.

2) Residual protein binding sites are saturated by incubating the plate with 0.1% HSA-TBS blocking buffer (0.1% (w / v) HSA in 10 mM Tris-Cl, 140 mM NaCl 1.5 mM NaN 3, pH 7.4) for 1 to 3 hours.

3) Plates are washed in TBS / tw / Ca ++ (TBS with 0.05% Tween 20 and 5 mM CaCl2) and diluted BBS is added to the samples (4 mM barbital, 145 mM NaCl, 2 mM CaCl 2, 1 mM MgCl 2, pH 7.4). Reservoirs receiving buffer only are used as negative controls. A control set of serum samples obtained from wild type or MASP-2 - / - mice are depleted in Clq prior to testing. Clq-depleted mouse serum was prepared using protein A-linked Dynabeads (Dynal Biotech, Oslo, Norway) coated with rabbit anti-human Clq IgG (Dako, Glostrup, Denmark) according to the supplier's instructions.

4) Following overnight incubation at 4 ° C and another wash with converted TBS / tw / Ca ++, converted C3 is detected with a polyclonal C3canti-human Antibody (Dako A 062) diluted in TBS / tw / Ca2 at 1: 1000). The secondary antibody is goat anti-rabbit IgG (whole molecule) conjugated to alkaline phosphatase (Sigma Immunochemicals A-3812) diluted 1: 10,000 in TBS / tw / Ca ++. The presence of an alternate complement pathway (AP) is determined by the addition of 100 μl of substrate solution (Sigma Fast p-Nitrophenyl Phosphate Suite sets) and incubation at room temperature. Hydrolysis is quantitatively monitored by measuring absorption at 405 nm in a microtiter plate reader. A standard curve is prepared for each analysis using serial dilutions of plasma / serum samples.

Results: The results shown in FIGURES 7A and 7B are pooled serum from various mice. Crosses represent MASP-2 + / + serum, full circles represent MASP-2 + / + serum depleted in Clq, open squares represent MASP-2 - / - serum and open triangles represent MASP-2 - / - serum depleted in Clq . As shown in FIGURES 7A-B, serum from MASP-2 - / - mice tested on a C3b deposition assay results in very low levels of C3 activation in mannan (FIG. 7A) and zimosan (FIG. 7B) coated plates. Results clearly demonstrate that MASP-2 is required to contribute to the initial generation of C3b from C3 to initiate the alternative complement path. This is a surprising result from the widely held point of the widely accepted theory that complement factors C3, factor B, factor D, and properdin form an independent functional alternative pathway in that C3 can undergo a spontaneous conformational change to a "C3b-equivalent" form that then generates a fasefluid iC3Bb convertase and deposits C3b molecules on activation surfaces such as zymosan.

Recombinant MASP-2 Reconstitutes Caminhoda lectin-Dependent C4 Activation in MASP-2 Mouse Serum - / -

In order to establish that the absence of MASP-2 was the direct cause of lectin pathway-dependent loss of C4 activation in the MASP-2 - / - mice, the effect of adding recombinant MASP-2 protein to serum samples was examined in the C4 cleavage described above. Recombinant functionally active murine MASP-2 and catalytically inactive murine MASP-2A recombinant proteins (wherein the serine protease domain active site serine residue was replaced in place of the alanine residue) were produced and purified as described below in Example 5. Pooled sera from 4 MASP-2-I-mice were preincubated with increasing protein concentrations of recombinant murine MASP-2 or inactive recombinant murine MASP-2A and C4 convertase activity was assayed as described above.

Results: As shown in FIGURE 8, the addition of functionally active recombinant murine MASP-2 protein (shown as open triangles) to serum obtained from MASP-2 - / - mice restored the pathway of lectin-dependent C4 activation in a concentration-dependent manner. whereas catalytically inactive murine MASP-2A protein (shown as stars) does not restore C4 activation. The results shown in FIGURE 8 are normalized to the C4 activation observed with pooled wild type mouse serum (shown as a dotted line).

EXAMPLE 3

This example describes the generation of a mouse-strain strain that is MASP-2 - / -, murine MAp 19 + / + and expresses a human MASP-2 transgene (a dead murine MASP-2 and an operating human MASP-2) .

Materials and Methods: A human MASP-2 encoding minigene called "mini hMASP-2" (SEQ ID NO: 49) as shown in FIGURE 5 was constructed which includes the promoter region of the human MASP 2 gene, including the first 3 exons (exon 1 exon 3) followed by the cDNA sequence representing the coding sequence of the following 8 exons, thereby encoding the full-size MASP-2 protein directed by this endogenous promoter. The hMASP-2 mini-construct was injected into fertilized MASP-2 - / - eggs to replace the deficient murine MASP-2 gene with the transgenically expressed human MASP-2.

EXAMPLE 4

This example describes the isolation of the human MASP-2 protein in pro-enzyme form from human serum.

Isolation method of human MASP-2: A paraisolar method of MASP-2 from human serum has been described in Matsushita et al., J. Immunol. 165: 2637-2642, 2000. In summary, human serum is passed through a mannan-Sepharose yeast column using a 10 mM deimidazole buffer (pH 6.0) containing 0.2 M NaCl, 20 mM CaCl2, 0.2 mM NPGB, 20 μΜ ρ-APMSF and 2% mannitol. MASP-I eMASP-2 pro-enzymes complex with AlBL and elute with the above buffer containing 0.3 M mannose. To separate MASP-I and MASP-2 proenzymes from MBL, preparations containing the complex are applied to anti-MBL-Sepharose and then MASPs are eluted with imidazole buffer containing 20 mM EDTA and 1 M NaCl. Finally, the MASP-I and MASP-2 pro-enzymes are separated from each other by passing through the same buffer anti-MASP-I-Sepharose as used for the anti-MBL-Sepharose. MASP-2 is recovered in the effluents, while MASP-I is eluted with 0.1 M glycine buffer (pH 2.2).

EXAMPLE 5

This example describes recombinant expression and protein production of human-sized mouse, rat and murine MASP-2, recombinant, MASP-2-derived polypeptides, and catalytically inactivated mutant forms of MASP-2. Human MASP-2 expression, life-size murine and rat

The full-length human MASP-2 cDNA sequence (SEQ ID NO: 4) was also subcloned into the mammalopCI-Neo expression vector (Promega) 5 which directs eukaryotic expression under the control of enhancer / promoter region of CMV (described in Kaufman RJ et al., Nucleic Acids Research 19: 4485-90, 1991; Kaufman, Methods in Chemistry, 185: 537-66 (1991)). The size-matched mouse cDNA (SEQ ED NO: 50) and rat MASP-2 cDNA (SEQ ID NO: 53) were each subcloned into the pED expression vector. The MASP-2 expression vectors were then transfected into the adherent Chinese hamster devarian cell line DXB1 using the standard calcium phosphate transfection procedure described in Maniatis et al., 1989. Ascells transfected with these constructs grew very slowly, implying that encoded protease is cytotoxic.

In another method, the minigene construct (SEQ ID NO: 49) containing the human MASP-2 cDNA targeted by its promoterendogen is transiently transfected into hamsterchinese ovary (CHO) cells. Human MASP-2 protein is secreted into the isolated culture medium as described below.

Life-size catalytically inactive MASP-2 expression:

Rationale Analysis: MASP-2 is activated by auto-catalytic cleavage after MBL orficoline recognition subcomponents (L-ficoline, H-ficoline or M-ficoline) bind to their respective carbohydrate pattern. Autocatalytic cleavage resulting in MASP-2 activation often occurs during the MASP-2 serum isolation procedure or during subsequent purification of recombinant expression. In order to obtain a more stable protein preparation for use as an antigen, a catalytically inactive form of MASP-2, designed as MASP-2A was created by replacing the serine residue that is present in the protease domain catalytic triad with a rat alanine (SEQ ID NO: 55 Ser617 to Ala617); in mouse (SEQ ID NO: 52 Ser617a Ala617); or in human (SEQ ID NO: 3 Ser618 to Ala618).

In order to generate catalytically inactive emurine human MASP-2A proteins, site-directed mutagenesis was performed using the oligonucleotides shown in TABLE 5. The oligonucleotides in TABLE 5 were designed to anneal the human and murine docDNA region encoding the enzymatically reactive serine and oligonucleotide contain a mismatch to change the serine codon into an alanine codon. For example, oligonucleotides ofPCR SEQ ID NOS: 56-59 were used in combination with human deMASP-2 cDNA (SEQ ID NO: 4) to amplify the codon region divided for enzymatically active serine and serine for stop codon for generate the full open reading of mutated MASP-2A containing mutation from Ser618 to Ala618. PCR products were purified after agarose gel electrophoresis and strip preparation and single adenosine overlaps were generated using a standard tail deformation procedure. Adenosine-tailed MASP-2A was then cloned into the loose vector pGEM-T, transformed into E. coli.

A catalytically inactive ratophilus MASP-2A protein is generated by kinase and annealing SEQ ID NO: 64 and SEQ IDNO: 65 by combining these two oligonucleotides in molar-like amounts, heating at 100 ° C for 2 minutes and slowly cooling. to room temperature. The resulting annealed fragment has PstI and XbaI compatible ends and was inserted in place of the wild-type rat MASP-2 cDNA PstI-XbaIdo fragment (SEQ ID NO: 53) to generate rat MASP-2A.

5 'GAGGTGACGCAGGAGGGGCATTAGTGTTT 3' (SEQ DD NO: 64)

5 'CTAGAAACACTAATGCCCCTCCTGCGTCACCTCTGCA 3' (SEQ ID NO: 65)

Human, murine, and rat MASP-2A were each further subcloned into the pED or pCI-Neo mammalian expression vectors and transfected into the Chinese hamster ovary cell line DXB 1 as described below.

In another method, a catalytically inactive form of MASP-2 is constructed using the method described in Chen et al., J. Biol.Chem., 276 (28): 25894-25902, 2001. In summary, the ocDNA-containing plasmid of Life-size human MASP-2 (described in Thiel et al., Nature 386: 506, 1997) is digested with XhoI and EcoRI and the MASP-2 CDNA (described herein as SEQ ID NO: 4) is cloned into the sites. restrictions corresponding to the pFastBacl baculovirus transfer vector (LifeTechnologies, NY). The active serine protease site of MASP-2 in Ser618 is then changed to Ala618 by replacing the double stranded oligonucleotides encoding amino acids 610 to 625 of the peptide region (SEQID NO: 13) with amino acids 610 to 625 of the native region to create a life-size MASP-2 polypeptide with a proteaseinative domain. Expression Plasmid Construction Containing Human Masp-2-Derived Polypeptide Regions.

The following constructs are produced using the MASP-2 desinal peptide (residues 1 to 15 of SEQ ID NO: 5) to secrete multiple MASP-2 domains. A construct expressing the human daMASP-2 CUBI domain (SEQ ID NO: 8) is manufactured by PCR by amplifying the region encoding residues 1 through 121 of MASP-2 (SEQ ED NO: 6) (which corresponds to the terminal CUB1 domain N). A construct that expresses human MASP-2 CUBIEGF domain (SEQ ID NO: 9) is fabricated byPCR by amplifying the region encoding residues 1 through 166 of MASP-2 (SEQ ID NO: 6) (which corresponds to the CUBLEGF domain of terminal) A construct expressing the human MASP-2 CUBIEGFCUBII domain (SEQ ED NO: 10) is manufactured by PCR by amplifying the region that encodes residues 1 through 293 of MASP-2 (SEQ ID NO: 6) (corresponding to CUBIEGFCUBII domain from terminal N). The above domains are amplified by PCR using VentR polymerase and pBS-MASP-2 as a standard according to established PCR methods. The 5 'sense primer sequence (5'-CGGGATCCATGAGGCTGCTGACCCTC-3' SEQ ID NO: 34) introduces a BamHI restriction site (underlined) at the 5 'end of the PCR products. The antisense primers for each of the MASP-2 domains, shown below in TABLE 5, are designed to introduce a stop codon (bold) followed by an EcoRI site (underlined) at the end of each PCR product. Once amplified, the DNA fragments are digested with BamHI and EcoRI in the corresponding sites of the pFastBacl vector. The resulting constructs are characterized by restriction mapping and confirmed by dsDNA sequencing.

Table 5: MASP-2 PER initiators

<table> table see original document page 179 </column> </row> <table> <table> table see original document page 180 </column> </row> <table>

Recombinant eukaryotic expression of MASP-2 and the production of enzymatically inactive mouse, rat and human MASP-2A protein.

The above-described MASP-2 and MASP-2 expression constructs were transfected into DXB1 cells using the standard calcium phosphate transfection procedure (Maniatis et al., 1989). MASP-2 was produced in serum free medium to ensure that the preparations were not contaminated with other serum proteins. The medium was harvested from confluent cells every second day (four times in total). The recombinant deMASP-2A level produced on average approximately 1.5 mg / liter culture medium for each of the three species.

MASP-2 A protein purification: MASP-2A (Ser-Ala mutant described above) was purified by column affinity chromatography of MBP-A-agarose. This strategy allowed for rapid purification without the use of foreign labels. MASP-2A (100 to 200 ml of medium diluted with an equal volume of loading buffer (50 mM Tris-Cl, pH 7.5, containing 150 mM NaCl and 25 mM CaCl 2) was loaded onto an MBP affinity column. -ararose (4 ml) pre-equilibrated with 10 ml loading buffer Following washing with a further 10 ml loading buffer, the protein was eluted in 1 ml fractions of 50 mM Tris-Cl, pH 7.5 containing 1.25 M NaCl and 10 mM EDTA Fractions containing MASP-2A were identified by SDS-polyacrylamide gel electrophoresis where necessary, MASP-2A was further purified by trochanion chromatography on a MonoQ column (HR 5 (5) Protein was dialyzed with 50 mM Tris-Cl pH 7.5 containing 50 mM NaCl and loaded onto the equilibrated column in the same buffer Following washing, bound MASP-2A was eluted with a gradient of 0.05 to 1 M NaCl in 10 ml.

Results: Yields of 0.25 to 0.5 mg deMASP-2A protein were obtained from 200 ml medium. The molecular weight of 77.5 kDa determined by MALDI-MS is greater than the calculated unmodified polypeptide value (73.5 kDa) due to glycosylation. The binding of glycans at each of the N-glycosylation sites accounts for the observed mass. MASP-2A migrates as a single band on SDS-polyacrylamide gels, which demonstrate that it is not proteolytically processed during biosynthesis. The weighted average molecular weight determined by equilibrium ultracentrifugation is in accordance with the calculated value for homodimers of the glycosylated polypeptide.

PRODUCTION OF HUMANARECOMBINANT MASP-2 POLYPEPTIDES

Another method for producing recombinant MASP-2 and MASP2A-derived polypeptides is described in Thielens, N. M., et al., J. Immunol. 166: 5068-5077, 2001. Briefly, the insect cells of Spodoptera frugiperda (Sf9 Ready-Plaque cells obtained from Novagen, Madison, WI) are cultured and maintained in serum-free medium supplemented with 50 IU / L (Life Technologies). ml of penicillin and 50 mg / ml deestreptomycin (Life Technologies). Trichoplusia ni (High Five) insect cells (provided by Jadwiga Chroboczek, Institut de BiologieStructurale, Grenoble, France) are maintained on TC100 medium (LifeTechnologies) containing 10% FCS (Dominique Dutscher, Brumath5France) supplemented with 50 IU / ml penicillin and 50 mg / ml deestreptomycin. Recombinant baculoviruses are generated using the Bac-to-Bac system (Life Technologies). Bacmid DNA is purified using the Qiagen midiprep purification system (Qiagen) and is used to transfect Sf9 insect cells using cellfectin in Sf900 II SFM medium (LifeTechnologies) as described in the manufacturer's protocol. Recombinant viral particles are collected 4 days later, titrated by the placade virus assay and amplified as described by King and Posver in The Baculovirus Expression System: A Laboratory Guide, Chapman and Hall Ltd., London, pp.111-114, 1992.

High Five cells (1.75 x 10 10 cells / 175 cm long detained culture flask) are infected with recombinant viruses containing MASP-2 polypeptides in a multiplicity of infection of 2 in SF900 II SFM medium at 28 ° C for 96 h. Supernatants are collected by centrifugation and diisopropyl phosphorofluoridate is added to a final concentration of 1 mM.

MASP-2 polypeptides are secreted into the culture medium. Culture supernatants are dialyzed against 50 mM NaC 1.1 mM CaCl2, 50 mM triethanolamine hydrochloride, pH 8.1 and loaded at 1.5 ml / min on a Q-Sepharose Fast Flow column (Amersham PharmaciaBiotech) (2 Χ 12 cm) equilibrated in the same buffer. Elution is conducted by applying a 1.2 liter linear gradient to 350 mM NaCl in the same buffer. Fractions containing recombinant MASP-2 polypeptides are identified by Western blot analysis, precipitated by the addition of 60% (w / v) (NH 4) 2 SO 4 and left overnight at 4 ° C. The pellets are resuspended in 145 mM NaCl, 1 mM CaCl 2, 50 mM triethanolamine hydrochloride, pH 7.4 and applied to a balanced TSK G3000SWG (7.5 χ 600 mm) column (Tosohaas, Montgomeryville, PA). plug. The purified polypeptides are then concentrated to 0.3mg / ml by ultrafiltration on Microsep microconcentrators (ppm = 10,000) (Filtron, Karlstein, Germany). EXAMPLE 6

This example describes a method of producing polyclonal antibodies against MASP-2 polypeptides. Materials and Methods:

MASP-2 antigens: The anti-human MASP-2 polyclonal antiserum is produced by immunizing rabbits with the following isolated MASP-2 polypeptides: human MASP-2 (SEQID NO: 6) isolated from serum as described in Example 4; Recombinant human MASP-2 (SEQ ED NO: 6), MASP-2A containing the inactive protease domain (SEQ ID NO: 13), as described in Examples 4 and 5; and recombinant CUBI (SEQ ID NO: 8), CUBEGFI (SEQ IDNO: 9) and CUBEGFCUBII (SEQ ID NO: 10) expressed as described above in Example 5.

Polyclonal Antibodies: Six-week-old rabbits, initiated with BCG (Calmette-Guerin Bacillus Vaccine) are immunized by injecting 100 µl of 100 µμ / µl MASP-2 polypeptide into sterile saline solution. Injections are made every 4 weeks, with the antibody titer monitored by the ELISA assay as described in Example 7. Culture supernatants are collected for antibody purification by protein A affinity chromatography.

EXAMPLE 7

This example describes a method for producing mouse monoclonal antibodies against mouse or human MASP-2 polypeptides.

Materials and methods:

Male A / J mice (Harlan, Houston, Tex.), 8-12 weeks old, are subcutaneously injected with 100 μg of rMASP-2 or rMASP-2A human or rat polypeptides (manufactured as described in Example 4 or Example 5) in Freundcomplete's adjuvant (Difco Laboratories, Detroit, Mich.) in 200 μΐ phosphate buffered saline (PBS) pH 7.4. At two-week intervals the mice are twice subcutaneously injected with 50 μg of rMASP-2 or rMASP-2A human or rat polypeptide in incomplete Freund's adjuvant. In the fourth week the mice are injected with 50μg of human or rat rMASP-2 or rMASP-2A polypeptide in PBS and fused 4 days later.For each fusion, single cell suspensions are prepared from the spleen of an immunized mouse. and used for fusion with Sp2 / 0 myeloma cells. 5 x 10 5 of Sp2 / 0 and 5 χ 10 of spleen cells are fused in a medium containing 50% polyethylene glycol (PM 1450) (Kodak, Rochester, NY) and 5% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.). The cells are then adjusted to a concentration of 1.5 χ 10 ^ 5 spleen cells per 200 μl of the suspension in Iscove medium (Gibco, Grand Island, NY), supplemented with 10% fetal bovine serum, 100 units / ml penicillin, 100 μL / ml streptomycin, 0.1 mM dehypoxanthine, 0.4 μΜ aminopterin and 16 μΜ thymidine. Two hundred microliters of the cell suspension is added to each reservoir about twenty 96-well microculture plates. After about ten days the culture supernatants are screened for reactivity with purified MASP-2 factor in an ELISA assay.

ELISA Assay: Immulon 2 microtest plate wells (Dynatech Laboratories, Chantilly, Va.) Are coated by the addition of 50 μl of purified 50 ng / ml hMASP-2 or rat rMASP-2 (or rMASP-2A) overnight. at room temperature. The low concentration of MASP-2 for coating allows selection of high affinity antibodies.

After the coating solution is removed by tapping the plate, 200 μl of BLOTTO (skimmed milk powder) in PBS is added to the reservoir for one hour to block non-specific sites. One hour later, the reservoirs are then washed with a PBST buffer (PBS containing 0.05% Tween 20). Fifty microliters of culture supernatants from each fusion reservoir are collected and mixed with 50 μΐ of BLOTTO and then added to the individual reservoirs of the demotest plates. After one hour of incubation, the reservoirs are flushed with PBST. Bound murine antibodies are then detected with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Fc-specific) (Jackson ImmunoResearch Laboratories, WestGrove, Pa.) And diluted 1: 2,000 in BLOTTO. A peroxidase substrate solution containing 0.1% 3,3,5,5 tetramethyl benzidine (Sigma, St. Louis, Mo.) And 0.0003% hydrogen peroxide (Sigma) is added to the color development reservoirs. for 30 minutes. The reaction is terminated by the addition of 50 μl 2 M H2SO4 per well. Optical Density at 450 nm of the reaction mixture is read with a BioTek ELISA Reader (BioTek Instruments, Winooski, Vt.).

MASP-2 Binding Assay:

Culture supernatants that test positive in the MASP-2 ELISA assay described above can be tested in a binding assay to determine the binding affinity that MASP-2 inhibiting agents have for MASP-2. A similar assay can also be used to determine whether inhibitory agents bind to other antigens in the complement system.

Polystyrene microtiter plate wells (96-well media binding plates, Corning Costar, Cambridge, MA) are coated with MASP-2 (20 ng / 100 μl / well, Advanced Research Technology, San Diego, CA) in buffered saline with phosphate (PBS) pH 7.4 overnight at 4 ° C. After aspirating the MASP-2 solution, the reservoirs are blocked with PBS containing 1% Sericabovine Albumin (BSA; Sigma Chemical) for 2 hours at room temperature. MASP-2 uncoated reservoirs serve as the deep controls. Aliquots of hybridoma or anti-MASP-2 purified MoAbs supernatants at varying concentrations in blocking solution are added to the reservoirs. Following a 2 hour incubation at room temperature, the reservoirs are extensively rinsed with PBS. MASP-2-bound MASP-2 anti-MoAb is detected by the addition of peroxidase-conjugated goat anti-mouse IgG (Sigma Chemical) in blocking solution, which is allowed to incubate for 1 hour at room temperature. The plate is rinsed again carefully with PBS and 100μl of 3,3 ', 5,5'-tetramethyl benzidine (TMB) substrate (Kirkegaard and PerryLaboratories, Gaithersburg, MD) is added. The TMB reaction is quenched by the addition of 100 μl of phosphoric acid. The plate is read at 450 nm in a microplate reader (SPECTRA MAX 250, Molecular Devices, Sunnyvale, CA).

Positive reservoir culture supernatants are then tested for the ability to inhibit complement activation in a functional assay such as the C4 cleavage assay as described in Example 2. The cells in the positive reservoirs are then cloned by limiting dilution. MoAbs are tested again for reactivity with hMASP-2 in an ELISA as described above. The selected hybrids are cultured in spinner flasks and the culturagas supernatant collected for antibody purification by protein A affinity chromatography.

EXAMPLE 8

This example describes the generation of a MASP-2 - / - -silenced mouse expressing human MASP-2 for use as a model in which to screen for MASP-2 inhibiting agents.

Materials and Methods: A MASP-2 - / - mouse as described in Example 1 and a MASP-2 - / - mouse expressing a human MASP-2 (operant human MASP-2) transgene construct as described in Example 3 are crossed and progenies which are murine25 MASP-2 - / -, murine MAp 19+, human MASP-2 + are used to identify human MASP-2 inhibitors.

Such animal models may be used as test substrates for the identification and efficacy of MASP-2 inhibiting agents such as human anti-MASP-2 antibodies, MASP-2 and non-peptide inhibiting peptides and compositions comprising MASP-2 inhibiting agents. 2. For example, the animal model is exposed to a compound or agent that is known to trigger MASP-2 dependent complement activation and a MASP-2 inhibitory agent is administered to the animal model at a time and concentration sufficient to evoke a reduction in symptoms. disease in the exposed animal.

In addition, murine MASP-2 - / -, MAp 19 +, human MASP-2 + mice can be used to generate cell lines containing one or more cell types involved in a MASP-2-associated disease. used as a model of cell culture for that disorder. Generation of continuous cell lines from transgenic animals is well known in the art, for example see Small, J.A., et al., Mol. Cell Biol., 5: 642-48, 1985.

EXAMPLE 9

This example describes a method of producing antibodies

against human MASP-2 in a MASP-2 silenced mouse expressing human MASP-2 and human immunoglobulins.

Materials and methods:

A MASP-2 - / - mouse was generated as described in Example 1. A mouse was then constructed that expresses human MASP-2 as described in Example 3. A homozygous MASP-2 - / - mouse and a MASP-2 - / - mouse that expresses human MASP-2 is crossed with a mouse derived from an embryonic stem cell line engineered to contain targeted disruptions of endogenous immunoglobulin heavy chain and light chain sites and expression by at least one segment of the human immunoglobulin site. of the human immunoglobulin site includes non-rearranged sequences of heavy and light chain components. Both inactivation of endogenous immunoglobulin genes and the introduction of degenerate exogenous immunoglobulin can be achieved by targeted homologous recombination. The transgenic mammals that result from these processes are capable of functionally rearranging the immunoglobulin component sequences and expressing a repertoire of antibodies of various isotypes encoded by the human immunoglobulin genes without expressing endogenous immunoglobulin genes.

The production and properties of mammals having these properties are described, for example see Thomson, AD, Nature 148: 1547-1553, 1994 and Sloane, BF, Nature Biotechnology 14: 826, 1996. Mice are inactivated and functionally substituted with human antibody genes are commercially available (eg, XenoMousP, available from Abgenix, Fremont CA). The resulting progeny mice are capable of producing human MoAb against human MASP-2 that are suitable for use in human therapy.

EXAMPLE 10

This example describes the generation and production of humanized murine anti-MASP-2 antibodies and antibody fragments.

A murine monoclonal anti-MASP-2 antibody is generated in male AU mice as described in Example 7. The demurine antibody is then humanized as described below to reduce its immunogenicity by replacing the murine constant regions with their human counterparts to generate a chimeric IgG and Antibody Fab fragment, which are useful for inhibiting the adverse effects of MASP-2-dependent complement activation in human patients according to the present invention.

1. Cloning of anti-MASP-2 variable region genes from murine hybridoma cells. Total RNA is isolated from anti-MASP-2 MoAb secreting hybridoma cells (obtained as described in Example 7) using RNAzole following the manufacturer's protocol (Biotech, Houston, Tex.). The first strand cDNA is synthesized from totalus oligo dT RNA as the primer. PCR is performed using the immunoglobulin constant C-region derived 3 'primers and degenerating primer sets derived from the leader peptide or the first murine Vh or Vk degene matrix region as the 5' primers. Anchored PCR is performed as described by Chen and Platsucas (Chen, P.F., Scand. J. Immunol.35: 539-549, 1992). For cloning of the VK gene, double stranded cDNA is prepared using a NotI-MAK1 primer (5'-TGCGGCCGCTGTAGGTGCTGTCTTT-3 'SEQ ID NO: 38). AD1 (5'-GGAATTCACTCGTTATTCTCGGA-3 'SEQ ID NO: 39) and AD2 (5' -TCCGAGAATAACGAGTG-3 'SEQ ID NO: 40) annealed adapters are attached to both the 5' and 3 'terminal of the double stranded cDNA. Adapters at the 3 'ends are removed by NotI digestion. The digested product is then used as the standard in PCR with oligonucleotide AD1 as the 5 'and MAK2 primer (5'-CATTGAAAGCTTTGGGGTAGAAGTTGTTC-3'SEQ ID NO: 41) as the 3' primer. Approximately 500 base pair DNA fragments are cloned into pUC19. Several clonons are selected for sequence analysis to verify that the cloned sequence covers the expected murine immunoglobulin constant region. NotI-MAK1 and MAK2 oligonucleotides are derived from the Vk region and are182 and 84 base pairs, respectively, downstream of the first pair. basic dogene C cover. Clones are chosen that include the full length Vk and leader opeptide.

For cloning of the VH gene, the double stranded cDNA is prepared using the NotI MAG1 primer (5'-CGCGGCCGCAGCTGCTCAGAGTGTAGA-3 'SEQ ID NO: 42). Annealed AD1 and AD2 adapters are attached to both the 5 'and 3' terminals of the double-stranded cDNA. The adapters at the 3 'ends are removed by NotI digestion. The digested product is used as standard in PCR with the AD1 and MAG2 oligonucleotide (5 '-CGGTAAGCTTCACTGGCTCAGGGAAATA-3' SEQ ID NO: 43) as primers. DNA fragments of 500 to 600 base pairs in length are cloned into pUC19. Not1-MAG1 and MAG2 oligonucleotides are derived from the murine C7.7.1 region and are 180 and 93 base pairs, respectively, downstream of the first base pair of the murine C7.7.1 gene. Clones are chosen that span the Vh peptide and full leader.

2. Construction of Expression Vectors for Chimeric deMASP-2 IgG and Fab. The cloned Vh and Vk genes described above are used as standard in a PCR reaction to add the Kozak consensus sequence to the 5 'end and the junction donor to the 3' end of the nucleotide sequence. After the sequences are analyzed to confirm the absence of PCR errors, the Vh and Vk genes are inserted into human C.71 and C. kappa expression vector cassettes respectively, to pSV2neoVH-huCy 1 and pSV2neoV-huCy. CsCl gradient purified plasmid DNAs from heavy and light chain vectors are used to transfect COS cells by electroporation. After 48 hours, the culture supernatant is tested by ELISA to confirm the presence of approximately 200 ng / ml chimeric IgG. Cells are harvested and total oRNA is prepared. First strand cDNA is synthesized from total RNA using oligo dT as the primer. This cDNA is used as the naPCR standard to generate the Fd and kappa DNA fragments. For the Fd gene, PCR is performed using 5'-AAGAAGCTTGCCGCCACCATGGATT GGCTGTGGAAC T-3 '(SEQ ID NO: 44) as the 5' primer and a 3 'primer derived from CH1 (5'-CGGGATCCTCAAACTTTCTTGTCCACTTGG-45') . The DNA sequence is confirmed to contain the complete Vh and CH1 domain of human IgG1.

After digestion with the appropriate enzymes, DNA fragments Fd are inserted into the HindIII and BamHI restriction sites of the pSV2dhfr-TUS expression vector cassette to give pSV2dhfrFd. The pSV2 plasmid is commercially available and consists of DNA segments from various sources: pBR322 DNA (thin line) contains the pBR322 origin of DNA replication (pBR ori) and the ampicillinalactamase resistance (Amp) gene; SV40 DNA, represented by the most striking shading, contains the SV40 origin of DNA replication (SV40 ori), promotorinitial (5 'to dhfr and neo genes) and polyadenylation signal (3' to dhfr and neo genes). The SV40-derived polyadenylation signal (pA) is also placed at the 3 'end of the Fd gene.

For the kappa gene, PCR is performed using 5'-AAGAAAGCTTGCCGCCACCATGTTCTCACTAGCTCT-3 '(SEQ ID NO: 46) as the 5' primer and a CK-derived 3 'primer (5'-CGGGATCCTTCTCCCTCTAACACTCT-3' SEQ ID NO: 47) . The DNA sequence is confirmed to contain the complete Vk and human CK regions. Following digestion with appropriate restriction enzymes, the capasin DNA fragments are inserted into the HindIII and BamHI restriction sites of the pSV2neo-TUS expression vector cassette to give pSV2neoK. Both Fd and kappa gene expression are driven by the enhancers and promoters derived from HCMV. Since the Fd gene does not include the cysteine amino acid residue involved in interchain disulfide bonding, this chimeric recombinant Fab contains noncovalently linked heavy and light chains. This chimericFab is referred to as cFab.

To obtain recombinant Fab with a heavy and light interchain disulfide bond, the above Fd gene may be extended to include an additional 9 amino acid coding sequence (EPKS CDKTH SEQID NO: 48) from the human IgG1 hinge region. The BstEII-BamHI DNA segment encoding 30 amino acids at the 3 'end of the Fd gene can be replaced with extended Fd encoding DNA segments, resulting in pSV2dhfrFd / 9aa.

3. Chimeric Anti-MASP-2 IgG Expression and Purification

To generate anti-MASP-2 chimeric IgG secreting cell lines, NSO cells are transfected with pSV2neoVH-huCyl and pSV2neoV-huC purified plasmid DNAs by electroporation. The transfected cells are selected in the presence of 0.7 mg / ml G418. The cells are grown in a 250 ml spinner flask using serum-containing medium.

The 100 ml spin culture culture supernatant is loaded onto a 10 ml PROSEP-A column (Bioprocessing, Inc., Princeton, N.J.). The column is washed with 10 bed volumes of PBS. Bound antibody is eluted with 50 mM citrate buffer, pH 3.0. Equal volume of 1M Hepes, pH 8.0 is added to the fraction containing purified antibody to adjust pH to 7.0. Residual salts are removed by exchanging buffer with PBS for Millipore membrane ultrafiltration (M.P. cut: 3,000). Protein concentration of the purified antibody is determined by the BCA method (Pierce).

4. Expression and purification of chimeric anti-MASP-2 Fab

To generate anti-MASP-2 chimeric Fab secreting cell lines, CHO cells are transfected with purified plasmid DNAs from pSV2dhfrFd (or pSV2dhfrFd / 9aa) and pSV2neocapa by electroporation. Transfected cells are selected in the presence of G418 and methotrexate. Selected cell lines are amplified at increasing concentrations of methotrexate. Cells are single cell subcloned by limiting dilution. High-producing single cell subcloned cell lines are then cultured in 100 ml spin culture using serum-free medium.

Chimeric anti-MASP-2 Fab is purified by affinity chromatography using a mouse anti-idiotypic MoAb for MASP-2 MoAb. An anti-idiotypic MASP-2 MoAb can be manufactured by immunizing mice with a keyhole limpet hemocyanin (KLH) murine conjugated MASP-2 anti-MoAb and screening for specific MoAb binding that can be competed with MASP-2. 2human. For purification, 100 ml of CHO cell culture spinner supernatant producing cFab or cFab / 9aa is loaded onto the affinity column bound with an anti-idiotypic MASP-2 MoAb. The column is then carefully washed with PBS before the bound Fab is eluted with 50mM diethylamine, pH 11.5. Residual salts are removed by buffer exchange as described above. Protein concentration of purified Fab is determined by the BCA method (Pierce).

The ability of chimeric MASP-2 IgG, cFab and cFAb / 9a to inhibit MASP-2 dependent complement pathways can be determined using the inhibitory assays described in Example 2.

EXAMPLE 11

This example describes an in vitro vitrous C4 cleavage assay as a functional screening to identify MASP-2 inhibitory agents capable of blocking MASP-2 dependent complement activation via L-ficoline / P35, H-ficoline, M-ficoline or Manana.

C4 Cleavage Assay: A C4 cleavage assay was described by Petersen, S. V., et al., J. Immunol. Methods 257: 107, 2001, who measured the activation of the lectin pathway resulting from L-ficoline-binding S. aureus lipoteichoic acid (LTA).

Reagents: Formalin fixed S. aureous (DSM20233) is prepared as follows: Bacteria are grown overnight at 37 ° C in tryptic soybean blood, washed three times with PBS, then fixed for 1 hour at room temperature in PBS / 0, 5% formalin and washed three more times with PBS before being resuspended in coating buffer (15 mM Na 2 CO 3, 35 mM NaHCO 3, pH 9.6).

Assay: The wells of a NuncMaxiSorb microtiter plate (Nalgene Nunc International, Rochester, NY) are coated with: 100 μΐ DSM20233 formalin-fixed S. aureus (OD550 = 0.5) in 1 μg L-ficoline coating buffer in coating buffer. After night incubation, the reservoirs are blocked with 0.1% human albumin (HSA) in TBS (10 mM Tris-HCl, 140 mM NaCl, pH 7.4), then washed with TBS containing 0.05% Tween 20 and 5 mM CaCl 2 (wash buffer). Human serum samples are diluted in 20mM Tris-HCl, 1 M NaCl, 10 mM CaCl2, 0.05% Triton X-100.0.1% HSA, pH 7.4, which prevents activation of Endogenous C4 and dissociates the Cl complex (composed of Clq, Clr and Cls). MASP-2 inhibiting agents, including MASP-2 anti-MoAbs and inhibiting peptides are added to serum samples at varying concentrations. Diluted samples are added to the plate and incubated overnight at 4 ° C. After 24 hours, the plates are washed thoroughly with wash buffer, then 0.1 μg purified human C4 (obtained as described in Dodds, AW, MethodsEnzymol. 223: 46, 1993) in 100 μl 4 mM barbital, 145 mM NaCl, 2mM CaCl2, 1mM MgCl2, pH 7.4 is added to each reservoir. After 1.5 hours at 37 ° C, the plates are washed again and C4b deposition is detected using anti-C4. chicken human alkaline phosphatase conjugate (obtained from Immunsystem, Uppsala, Sweden) and measuring the colorimetric substrate of p-nitrophenyl phosphate.

Mannan C4 Assay: The assay described above is adapted to measure lectin pathway activation by MBL by plating the plate with LSP and mannan prior to the addition of mixed serum with various MASP-2 inhibiting agents.

H-Ficoline C4 Assay (Hakata Ag): The assay described above is adapted to measure lectin pathway activation by H-ficoline by plating the plate with LPS and H-ficoline prior to the addition of desorus mixed with various inhibiting agents. of MASP-2.EXAMPLE 12

The following assay demonstrates the presence of classic pathway activation in wild type and MASP-2 - / - mice.

Methods: Immune complexes were generated in situ by coating microtiter plates (Maxisorb5 Nunc, cat. No. 442404, Fisher Scientific) with 0.1% human serum albumin in 10 mM Tris, 140 mM NaCl, pH 7.4 per ml. 1 hour at room temperature followed by overnight incubation at 4 ° C with whole sheep antiserum antiserum (Scottish Antibody ProductionUnit, Carluke, Scotland) diluted 1: 1000 in TBS / tween / Ca. Desorption samples were obtained from wild type and MASP-2 - / - mice added to the coated plates. Control samples were prepared where Clq was depleted from wild-type and MASP-2 - / - serum samples Clq-depleted mouse serum was prepared using IgG-coated protein A Dynabeads (Dynal Biotech, Oslo, Norway). rabbit anti-Clq (Dako, Glostrup, Denmark) according to the supplier's instructions. Plates were incubated for 90 minutes at 37 ° C. Bound C3b was detected with a polyclonal human anti-C3c antibody (Dako A 062) diluted in 1: 1000 TBS / tw / Ca2. The secondary antibody is goat anti-rabbit IgG. Results: FIGURE 9 shows the relative deposition of

C3b levels in IgG coated plates in wild-type serum, MASMAS-2 - / - serum, Clq-depleted wild-type serum and Clq-depleted MASP-2 - / - serum. These results demonstrate that the classical pathway is intact in the MASP-2 - / - mouse strain.

The following assay is used to test whether a MASP-2 inhibitory agent blocks the classical pathway by analyzing the effect of a MASP-2 inhibitory agent under conditions where the classical pathway is initiated by immune complexes. Methods: To test the effect of a MASP-2 inhibitory agent under complement activation conditions where the classical pathway is initiated by immune complexes, 50 μΐ triplicate samples containing 90% NHS are incubated at 37 ° C in the presence of 10 μ ^ πιΐ immune complex (IC) or PBS and parallel triplicate (+/- IC) samples are also included that contain 200 nM anti-properdin monoclonal antibody during incubation at 37 ° C. After a two hour incubation at 37 ° C, 13 mM EDTA is added to all samples to stop further complement activation and the samples are immediately cooled to 5 ° C. Samples are then stored at -70 ° C prior to assaying for complement activation products (C3a and sC5b-9) using kitsELISA (Quidel, Catalog No. AO15 and A009) following the manufacturer's instructions.

EXAMPLE 14

This example demonstrates that the lectin-dependent MASP-2 complement-activation system is activated in the deischemia / reperfusion phase following repair of abdominal aortic aneurysm.

Experimental Rational Analysis and Planning: Patients undergoing abdominal aortic aneurysm repair (AAA) undergo an ischemia-reperfusion injury, which is largely mediated by complement activation. We investigated the role of the MASP-2 lectin-dependent pathway of complement activation in empathic ischemia-reperfusion injury undergoing AAA repair. Serum binding-binding lectin (MBL) consumption was used to measure the amount of MASP-2-dependent activation of the lectin pathway that occurred during perfusion.

Patient Serum Isolation: A total of 23 patients undergoing elective infrarenal AAA repair and 8 control patients undergoing major abdominal surgery were included in this study.

For patients undergoing AAA repair, systemic blood samples were taken from each patient's radial artery (through an arterial line) at four defined time points during the procedure: time point 1: induction of anesthesia; time point 2: just before aortic clamping; time point 3: exactly before aortic clamp removal; and time point 4: during the profile.

For control patients undergoing major abdominal surgery, systemic blood samples were taken for induction of anesthesia and within two hours of the start of the procedure.

MBL Levels Assay: Each patient plasma sample was assayed for mannan binding lectin (MBL) levels using ELISA techniques.

Results: The results of this study are shown in FIGURE 10, which presents a graph showing the average percentage change in MBL levels (y axis) at each of the various time points (x axis). The starting values for MBL are 100%, with the relative decreases shown below. As shown in FIGURE 10, AAA patients (n = 23) show a significant decrease in plasma MBL levels, producing an average 41% decrease approximately at the time of ischemia / reperfusion following AAA. In contrast, in control patients (n = 8) undergoing major abdominal surgery only lower MBL consumption was observed in plasma samples.

The data presented provide a strong indication that the MASP-2 dependent lectin pathway of the complement system is activated in the ischemia / reperfusion phase following AAA repair. Decreased MBL levels appear to be associated with deischemia-reperfusion injury because MBL levels drop significantly and rapidly when the clamped main vessel is reperfused after the end of the operation. In contrast, control sera from patients undergoing major abdominal surgery without a major ischemia-reperfusion insult only show a slight decrease in MBL plasma levels. In a well-established contribution of complement activation in perfusion injury, we conclude that activation of the MASP-2 lectin-dependent pathway in ischemic endothelial cells is a major factor in ischemia / reperfusion injury pathology. Therefore, a specific MASP-2-dependent transient blockade or reduction in the complement activation activation pathway would be expected to have a significant beneficial therapeutic impact to improve outcome of clinical procedures and diseases involving a transient ischemic insult, for example, myocardial infarction, myocardial infarction. bowel, burns, transplantation and stroke.

EXAMPLE 15

This example describes the use of the MASP-2 - / - strain as an animal model for testing MASP-2 inhibiting agents useful for treating Rheumatoid Arthritis.

Background and Rational Analysis: Murine Arthritis Model: K / BxN T cell receptor (Tg) transgenic mice (Tg) are a newly developed model of inflammatory arthritis (Kouskoff, V., et al, Cell 87: 811-822 , 1996; Korganow, AS, et al., Immunity 10: 451-461, 1999; Matsumoto, I., et al., Science 286: 1732-1735,1999; Maccioni M. et al., J. Exp. Med. 195 ( 8): 1071-1077, 2002). K / BxN mice spontaneously develop an autoimmune disease with most of the clinical, histological, and immunological features of RA in humans (Ji, H., et al., Immunity 16: 157-168, 2002). Demurine disorder is joint specific, but is then started perpetuated by T-cell reactivity, then B to glucose-6-phosphate isomerase ("GPI"), a ubiquitously expressed antigen. In addition, the transfer of serum (or purified anti-GPI IgG) from arthritic K / BxN mice to livestock causes arthritis within several days. Polyclonal anti-GPI antibodies or a pool of anti-GPI monoclonal antibodies to the isotype IgG1 have also been shown to induce arthritis when injected into healthy recipients (Maccioni et al., 2002). The murine model is relevant to human RA because serum from RA patients has also been found to contain anti-GPI antibodies, which is not found in normal subjects. A C5 deficient mouse was tested in this system and found to block the development of arthritis (Ji, H., et al., 2002, supra). There was also strong inhibition of arthritis in C3 null mice, implying the alternate pathway, however, MBP-A null mice do not develop marthritis. In mice however, the presence of MBP-C may compensate for the loss of MBP-A.

Based on the observations described here that MASP-2 plays an essential role in initiating both the alternative quantum lectin pathway, the K / BxN arthritic model is useful for screening for MASP-2 inhibitors that are effective for use as a therapeutic agent. to treat RA.

Methods: Serum from arthritic K / BxN mice is obtained at 60 days of age, pooled and injected (150 to 200 μΐ i.p.) into deMASP-2 - / - receptors (obtained as described in Example 1); and control litters with or without MASP-2 inhibiting agents (MoAb, inhibiting peptides and others as described herein) on days 0 and 2. A group of abnormal mice are also pretreated with a MASP-2 inhibiting agent two days before receiving the injection of serum. Another group of mice received a serum injection on day 0, followed by a DEMSP-2 inhibitory agent on day 6. A clinical index is evaluated over time with a point marked for each affected paw. . Ankle thickness is also measured by a caliber (thickness is defined as the difference in measurement from day 0). EXAMPLE 16

This example describes an assay for inhibition of complement-mediated tissue injury in an ex vivo model of human plasma perfused rabbit hearts.

Background and Rational Analysis: Activation of the complement system contributes to hyperacute rejection of xenografts. Previous studies have shown that hyperacute rejection can occur in the absence of anti-donor antibodies through alternative pathway activation (Johnston, P. S., et al., Transplant Proc. 23: 877-879, 1991).

Methods: To determine whether isolated anti-MASP-2 inhibiting agents such as anti-MASP-2 antibodies obtained as described in Example 7 are capable of inhibiting the complement pathway in the lesion detected, MASP-2 anti-MoAbs and antibody fragments can tested using an ex vivo model in which isolated rabbit hearts are infused with diluted human plasma. This model has previously been shown to cause rabbit myocardial injury due to activation of the alternative complement pathway (Gralinski, M. R., et al., Immunopharmacology34: 79-88, 1996). EXAMPLE 17

This example describes an assay that measures deneutrophil activation that is useful as a measure of an effective dose of a MASP-2 inhibitory agent for treating lectin-dependent pathway associated conditions according to the methods of the invention.

Methods: A method for measuring neutrophil elastase was described in Gupta-Bansal, R., et al., Molecular Immunol. 37: 191-201, 2000. In summary, the αΐ-antitrypsin serum elastase complex is measured with a two-site interleaved assay that uses antibodies to both elastase and α1-antitrypsin. Polystyrene microtiter plates are coated with a 1: 500 dilution of anti-human elastase antibody (TheBinding Site, Birmingham, UK) in PBS overnight at 4 ° C. After stripping the antibody solution, the reservoirs are blocked with PBS containing 0.4% TEM for 2 hours at room temperature. Aliquots (100 μl) of plasma samples that are treated with or without a MASP-2 inhibitor are added to the reservoirs. Following a 2 hour incubation at room temperature, the reservoirs are extensively rinsed with PBS. The elastase-al-antitrypsinigated complex is detected by the addition of a 1: 500 dilution of peroxidase-conjugated al-antitrypsin antibody in blocking solution which is allowed to incubate for 1 h at room temperature. After washing the comPBS plate, 100 μl aliquots of TMB substrate are added. The TMB reaction is quenched by the addition of 100 μl of phosphoric acid and the plate is read at 450nm in a microplate reader.

EXAMPLE 18

This example describes an animal model for testing MASP-2 inhibitors useful for treating myocardial ischemia / reperfusion.

Methods: A model of myocardial ischemia-reperfusion was described by Vakeva et al., Circulation 97: 2259-2267, 1998 and Jordan et al., Circulation 104 (12): 1413-1418, 2001. The described model can be modified for use. in MASP-2 - / - and MASP-2 + / + mice as follows. In summary, adult male mice are anesthetized. The vein and trachea are cannulated and ventilation is maintained with 100% oxygen with a rodent ventilator adjusted to keep exhaled CO2 between 3.5% and 5%. A left thoracotomy is performed and a suture is placed 3 to 4 mm from the origin of the left coronary artery. Five minutes before ischemia, the animals are administered a MASP-2 inhibitory agent, such as anti-MASP-2 antibodies (for example, in a fingertip range of 0.01 to 10 mg / kg). Ischemia is then initiated by tightening the suture around the coronary artery and maintained for 30 minutes, followed by four hours of reperfusion. Simulated operated animals are prepared individually without tightening the suture.

Complement Deposition Analysis C3: After perfusion, samples for immunohistochemistry are obtained from the central region of the left ventricle, fixed and frozen at -80 ° C until processed. Tissue sections are incubated with an HRP-cabraged C3 anti-mouse antibody. Tissue sections are analyzed for the presence of C3 dyeing in the presence of anti-MASP-2 inhibiting agents compared with sham operated control animals and MASP-2 - / - animals to identify MASP-2 inhibiting agents that reduce C3 deposition. in vivo.

Example 19

This example describes the use of the MASP-2 - / - strain as an animal model to test MASP-2 inhibiting agents for its ability to protect transplanted tissue from ischemia / reperfusion injury.

Background / Rational Analysis: It is known that the deischemia / reperfusion injury occurs in a donor organ during transplantation. Tissue injury rate is related to the extent of ischemia and is mediated by complement, as demonstrated in various models of ischemia and through the use of complement inhibitory agents such as soluble receptor type 1 (CRI) (Weisman et al., Science 249: 146 -151, 1990; Mulligan et al., J. Immunol. 148: 1479-1486 (1992; Pratt et al., Am. J. Path.163 (4): 1457-1465, 2003). An animal model for transplantation was described by Pratt et al., Am. J. Path. 163 (4): 1457-1465, which may be modified for use with the MASP-2 - / - mouse model and / or for use as a MASP-2 + / + model system in which to screen for MASP- 2 for the ability to protect transplanted tissue from ischemia / reperfusion injury. Flooding the donor kidney with perfusion fluid prior to transplantation provides an opportunity to introduce anti-MASP-2 inhibitor agents into the donor kidney.

Methods: MASP-2 - / - and / or MASP-2 + / + mice are anesthetized. The left donor kidney is dissected and the aorta is connected to the head and towards the tail to the renal artery. A tuboportex catheter (Portex Ltd5 Hythe, UK) is inserted between the bandages and the kidney is infused with 5 ml Soltran Renal Perfusion Solution (Baxter HealthCare, UK) containing MASP-2 inhibiting agents such as anti-MASP-2 monoclonal antibodies. (over a dosage range of 0.01 mg / kg to 10mg / kg) for a period of at least 5 minutes. The kidney transplant is then performed and the mice are monitored over time.

Transplant Recipient Analysis: Transplantation is harvested at various time intervals and tissue sections are analyzed using anti-C3 to determine the degree of C3 deposition.

EXAMPLE 20

This example describes the use of an animal collagen-induced arthritis (CLA) model to test MASP-2 inhibiting agents useful for treating rheumatoid arthritis (RA).

Background and Rational Analysis: Collagen-induced arthritis (CIA) represents an inducible autoimmune polyarthritis in rodent and primate cepsensitive after immunization with native type II collagen and is recognized as a relevant model for human rheumatoid arthritis (RA) (see Courtney et al. , Nature 283: 666 (1980); Trenthan et al., J.Exp. Med. 146: 857 (1977)). Both RA and ASD are characterized by joint inflammation, cloth formation, and cartilage and bone erosion. CIA-susceptible murine acepa DBA / ILacJ is a developed model of CIA in which mice develop clinically severe arthritis following immunization with bovine collagen type II (Wang et al., J. Immunol.164: 4340-4347 (2000). CS-deficient mouse strain strained with DBA / ILacJ and the resulting strain has been found to be resistant to the development of CIA arthritis (Wang et al., 2000, supra).

Based on the observations described herein that MASP-2 plays an essential role in initiating both the alternative quantum lectin pathway, the arthritic model of CIA is useful for screening for MASP-2 inhibitors that are effective for use as therapeutic agents for treating RA. .

Methods: A MASP-2 - / - mouse is generated as described. Example 1. The MASP-2 - / - mouse is then mated with a mouse derived from the DBA / ILacJ strain (The Jackson Laboratory). The progeny F1 and subsequent progeny are interbreeded to produce MASP-2 - / - homozygotes in the DBA / ILacJ strain.

Collagen immunization is performed as described in Wang et al., 2000, supra. In summary, wild type DBA / ILacJ and DBA / ILacJ MASP-2 - / - mice are immunized with bovine collagen type II (BCII) or mouse collagen type II (MCII) (obtained from Elastin Products, Owensville, MO), dissolved. in 0.01 M acetic acid at a concentration of 4 mg / ml. Each mouse is injected intradermally at the base of the tail with 200 μg CII and 100 μg demicobacteria. The mice are reimmunized after 21 days and are examined daily for arthritis. An arthritic index is evaluated over time with respect to the severity of affected arthritis in the patient.

MASP-2 inhibitory agents are screened in wild-type DBA / ILacJ CIA mice by injection of a MASP-2 inhibitor such as anti-MASP-2 monoclonal antibodies (within a dose range of 0.01 mg / kg to 10 mg / kg) at the time of immunization with collagen, systemically or locally in one or more joints and a tritic index is evaluated over time as described above. Anti-hMASP-2 monoclonal antibodies as therapeutic agents can be easily evaluated in a DBA / ILacJ CIA MASP-2 - / -, hMASP - + / + operant mouse model.

EXAMPLE 21

This example describes the use of an animal model (NZB / W) F1 to test MASP-2 inhibitory agents useful for treating immune complex mediated agglomerulonephritis.

Background and Rational Analysis: New Zealand black χ New Zealand black mice (NZB / W) Fl spontaneously develop an autoimmune syndrome with striking similarities to human immune complex glomerulonephritis. NZB / W mice Flinvariably succumb to glomerulonephritis at 12 months of age. As discussed above, complement activation has been shown to play a significant role in the pathogenesis of immune complex-mediated glomerulonephritis. Administration of an anti-C5 MoAb to the NZB / W Fl mouse model has also been shown to result in significant improvement in the course of glomerulonephritis (Wang et al., Proc. Natl. Acad. Sci. 93: 8563-8568 (1996)) . Based on the observations described herein that MASP-2 plays an essential role in initiating both the lectin and alternative pathways, the NZB / W Fl animal model is useful for screening for MASP-2 inhibiting agents that are effective for use as therapeutic agents to treat glomerulonephritis.

Methods: A MASP-2 - / - mouse is generated as described. Example 1. The MASP-2 - / - mouse is then separately mated with a mouse derived from both NZB and NZW (TheJackson Laboratory). Fl and subsequent progeny are intertwined to produce MASP-2 - / - homozygotes on both the NZB and NZW quantone genetic basis. To determine the role of MASP-2 in the pathogenesis of daglomerulonephritis in this model, the development of this disease in F1 individuals resulting from crosses of NZB x NZW wild type mice or MASP-2 - / - NZB x MASP-2 - / - NZW mice is compared. At weekly intervals urine samples will be collected from F1 MASP-2 + / + and MASP-2 - / - mice and urine protein levels monitored for the presence of anti-DNA antibodies (as described in Wang et al., 1996, supra). . Histopathological analysis of the kidneys is also performed to monitor the amount of mesangial matrix deposition and glomerulonephritis development.

The NZB / W F1 animal model is also useful for screening for MASP-2 inhibiting agents that are effective for use as a therapeutic agent to treat glomerulonephritis. At 18 weeks of age, wild-type NZB / W F1 mice are injected intraperitoneally with anti-MASP-2 inhibiting agents, such as monoclonal antibodies -anti-MASP-2 (over a dose range of 0.01 mg / kg to 10 mg). / kg) at weekly or bi-weekly frequency. The above mentioned biochemical histopathological markers of glomerulonephritis are used to assess the development of the disease in mice and to identify MASP-2 inhibitors useful for the treatment of this disease.

EXAMPLE 22

This example describes the use of a tube loop as a model for testing MASP-2 inhibitory agents useful for preventing sustained injury resulting from cardiopulmonary bypass (ECC) such as a cardiopulmonary bypass circuit (CPB).

Rationale and Rationale: As discussed above, patients who undergo ECC during CPB suffer from a systemic inflammatory reaction, which is partly caused by exposure of the blood to artificial surfaces of the extracorporeal circuit, but also by independent surface factors such as surgical trauma and ischemic injury. reperfusion (Butler J., et al., Ann. Thorac. Surg. 55: 552-9, 1993; Edmunds, LH, Ann. Thorac. Surg. 66 (Suppl): S12-6, 1998; Asimakopoulos, G., Perfiision 14: 269-77, 1999). The alternative decompression pathway has also been shown to play a predominant role in the activation of decompression in CPB circuits, which results from the interaction of blood with artificial surfaces of the CPB circuits (see Kirklin et al., 1983, 1986, discussed above). Therefore, based on the observations described here that MASP-2 plays an essential role in initiating both the dalectin and alternative pathways, the tube loop model is useful for screening for MASP-2 inhibiting agents that are effective for use. as therapeutic agents to prevent or treat an inflammatory reaction triggered by extracorporeal exposure.

Methods: A modification of a previously described tube loop model for cardiopulmonary bypass circuits is used (see Gong et al., J. Clinic Immunol. 16 (4): 222-229 (1996)) as described in Group-Bansal et al. , Molecular Immunol. 37: 191-201 (2000). In summary, blood is freshly collected from a healthy patient in a 7 ml vacutainer tube (containing 7 units of heparin per ml whole blood). Polyethylene tubing similar to that used during CPB procedures (eg, D.I. 2.92 mm; D.E. 3.73 mm, length: 45 cm) is filled with 1 ml of blood and closed in a loop with a short piece of desilicone tube. A control tube containing 10 mM heparinized EDTA blood was included in the study as a ground control. The sample and control tubes were rotated vertically in a 1 hour water bath at 37 ° C. After incubation, blood samples were transferred into 1.7 ml microcentrifuge tubes containing EDTA, resulting in a final concentration of 20 mM EDTA. The samples were centrifuged and the plasma was collected. MASP-2 inhibiting agents such as anti-MASP-2 antibodies are added to heparinized blood immediately prior to rotation. Plasma samples are subjected to assays for measuring the concentration of soluble C3a and C5b-9 as described in Gupta-Bansal et al., 2000, supra.

EXAMPLE 23

This example describes the use of a rodent cecal deletion and perforation (PLC) model system to test MASP-2 inhibitory agents useful for treating septicemia or a condition that results in septicemia, including severe septicemia, septic shock, acute respiratory distress syndrome of septicemia and systemic inflammatory response syndrome.

Background and Rational Analysis: As discussed above, complement activation has been shown in numerous studies to have a major role in the pathogenesis of septicemia (see Bone, R. C., Annals. Internai. Med. 115: 457-469, 1991). The CLP rodent model is a recognized model that mimics the clinical course of septicemia in humans and is considered to be a reasonable substitute model for septicemia in humans (see Ward, P., Nature Review Immunology Vol 4: 133-142 (2004). A recent study has shown that treatment of animals with PLC with anti-C5a antibodies has resulted in reduced bacteremia and greatly improved survival Huber-Lang et al., J. of Immunol. 169: 3223-3231 (2002). described herein that MASP-2 plays an essential role in initiating both the dalectin and alternative pathways, the CLP rodent model is useful for screening for MASP-2 inhibitors that are effective for use as therapeutic agents to prevent or treat septicemia or a condition resulting from septicemia.

Methods: The PLC model is adapted from the model described in Hub-Lang et al., 2004, supra as follows. Animals MASP-2 - / - and MASP-2 + 1 + are anesthetized. An abdominal incision in the 2 cm centerline is made and the caecum is firmly attached below the ileocecal valve, avoiding bowel obstruction. The caecum is then completely punctured with a 21 gauge needle. The abdominal incision was then layered with silk suture and skin clips (Ethicon, Summerville, NJ). Immediately after CLP, the animals receive an injection of a MASP inhibitor. -2 as anti-MASP-2 monoclonal antibodies (at a dosage range of 0.01 mg / kg to 10 mg / kg). Anti-hMASP-2 monoclonal antibodies as therapeutic agents can be readily evaluated in a mouse model of PLC operating on MASP-2 - / -, hMASP - + / +. Mouse plasma are then analyzed for complement-derived deanaphylatoxins and respiratory burst levels using the assays described in Huber-Lang et al., 2004, supra.

EXAMPLE 24

This example describes the identification of high affinity Fab2 anti-MASP-2 Fab2 antibody fragments that block deMASP-2 activity.

Background and Rational Analysis: MASP-2 is a complex protein with many separate functional domains, including: MBL and phytolines deletion site (s), a serine protease catalytic site, a binding site for the C2 proteolytic substrate, a binding site for C4 proteolytic substrate, one MASP-2 cleavage site for MASP-2 zymogen autoactivation and two Ca + "*" binding sites. Fab2 antibody fragments have been identified that bind with high affinity to MASP-2 and identified Fab2 fragments were tested in a functional assay to determine if they were able to block MASP-2 functional activity.

To block MASP-2 functional activity, an antibody or Fab2 antibody fragment must bind and interfere with a MASP-2 epitope that is required for MASP-2 functional activity. Therefore, many or all of the anti-MASP-Fab2s High affinity binding may not inhibit the functional activity of MASP-2 unless they bind the structural epitopes on MASP-2 that are directly involved in the functional activity of MASP-2.

A functional assay that measured inhibition of lectin C3 convertase pathway formation was used to evaluate the "blocking activity" of anti-MASP-2 Fab2s. It is known that the primary physiological role of MASP-2 in the lectin pathway is to generate the next functional component of the lectin mediated complement pathway, the seventh pathway of lectin C3 convertase. The lectin C3 convertase pathway is a critical enzyme complex (C4bC2a) that proteolytically cleaves C3 into C3a and C3b. MASP-2 is not a structural component of the lectinC3 convertase (C4bC2a) pathway; however, the functional activity of MASP-2 is required to generate the two protein components (C4b, C2a) that comprise the lectin C3 convertase pathway. In addition, all separate functional activities of MASP-2 listed above appear to be required so that MASP-2 manages the lectin C3 convertase pathway. For these reasons, it is believed that a preferred assay for use in evaluating Fab2s "blocking activity" anti-MASP-2 is a functional assay that measures inhibition of lectin C3 convertase pathway formation.

High Affinity Fab2s Generation: A phage demonstration library of human light and heavy chain antibody sequences and automated antibody selection technology to identify Fab2s that react with selected ligands of interest has been used to create high affinity Fab2s for the MASP protein. Rat -2 (SEQ ID NO: 55). A known amount of rat MASP-2 protein (~ 1 mg,> 85% pure) was used for antibody screening. Three rounds of amplification were used for antibody selection with improved fi nity. Approximately 250 different hits expressing antibody fragments were chosen for ELISA screening. High affinity hits were subsequently sequenced to determine the impairment of the different antibodies.

Fifty unique anti-MASP-2 antibodies were purified and 250 µg of each purified Fab2 antibody were used for characterization of MASP-2 binding affinity and functional pathway assay, as described in more detail below.

Assays Used to Evaluate Fab2s Anti-MASP-2 Inhibitor (Blocker) Activity

1. Assays for Measuring LectinConvertase Pathway Inhibition:

Background: The lectin C3 convertase pathway is the enzymatic complex (C4bC2a) that proteolytically cleaves C3 into the two potent proinflammatory fragments, anaphylatoxin C3a and opsonic C3b. C3 convertase formation appears to be a key step in the lectin pathway in terms of mediating inflammation. MASP-2 is not a structural component of the lectin C3 convertase (C4bC2a) pathway; therefore anti-MASP-2 (or Fab2) antibodies will not directly inhibit pre-existing C3 convertase activity. However, MASP-2 serine protease activity is required in order to generate the two protein components (C4b, C2a) which comprise the lectin C3 convertase pathway. Therefore, anti-MASP-2 Fab2 which inhibits the functional activity of MASP-2 (i.e. blocking anti-MASP-2 Fab2) will inhibit de novo formation of the lectin C3convertase pathway. C3 contains an unusual and highly reactive thioester group as part of its structure. On C3 cleavage by C3 convertase in this assay, the C3b thioester group can form a covalent bond with the hydroxyl or amino groups on the macromolecules immobilized at the bottom of the plastic reservoirs via ester or amide bonds, thereby facilitating the detection of C3b in the ELISA assay. .

Manana yeast is an activator of the known lectin pathway. In the following method to measure C3 convertase formation, the mannan-coated plastic reservoirs were incubated for 30 min at 37 ° C with diluted rat serum to activate the lectin pathway. The reservoirs were then washed and assayed for immobilized C3b in the reservoirs using standard ELISA methods. The amount of C3b generated in this assay is a direct reflection of de novo formation of the lectinC3 convertase pathway. Anti-MASP-2 Fab2s at selected concentrations were tested in this assay for their ability to inhibit C3convertase formation and consequent C3b generation.

Costar Medium Binding 96-well plates were incubated overnight at 50 ° C with manana diluted in 50 mM decarbonate buffer, pH 9.5 at 1 µg / 50 µl / well. After the night incubation, each reservoir was washed three times with 200 μl PBS. The reservoirs were then blocked with 100 μl / 1% serum albumin reservoir in PBS and incubated for one hour at room temperature with gentle mixing. Each reservoir was then washed three times with 200 μl of PBS. Anti-MASP-2 Fab2 samples were diluted to selected concentrations in GVB buffer containing Ca + * and Mg4 + + (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl2s 2.0 mM CaCl2, 0 , 1% gelatin, pH 7.4) at 5 ° C. A 0.5% rat serum was added to the above samples at 5 ° C and 100 µl were transferred to each reservoir. The plates were covered and incubated for 30 minutes in a 37 ° C water bath to allow complement activation. The reaction was stopped by transferring the plates from the 37 ° C water bath to a vessel containing an ice-water mixture. Each well was washed five times with 200 μl PBS-Tween 20 (0.05% Tween 20 in PBS), then washed twice with 200 μl PBS.A 100 μl / 1: 10,000 dilution reservoir of primary antibody ( rabbit anti-human C3c, DAKO A0062) was added in PBS containing 2.0 mg / ml bovine serum albumin and incubated 1 hour at room temperature with mild mixing. Each reservoir was washed 5 χ 200 μl PBS. 100μl / 1: 10,000 dilution reservoir of secondary antibody (peroxidase-conjugated goat anti-rabbit IgG, American Qualex A102PU) was added in PBS containing 2.0 mg / ml bovine serum albumin and incubated for one hour at room temperature on a shaker with smooth mix. Each chamber was washed five times with 200 μl of PBS. 100 μl / reservoir of TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 10 min. The peroxidase reaction was stopped by the addition of 100 μl / 1.0M H3PO4 reservoir and OD450 was measured.

2. Assay for Measuring PAS4-Dependent C4 Cleavage Inhibition

Background: MASP-2 serine protease activity is highly specific and only two MASP-2 protein substrates have been identified; C2 and C4. Cleavage of C4 yields C4a and C4b. Anti-MASP-2 Fab2 can bind to structural epitopes on MASP-2 that are directly involved in C4 cleavage (eg, MASP-2 paraC4 binding site; MASP-2 serine protease catalytic site) and thus inhibit activity C4 cleavage function of MASP-2.

Manana yeast is an activator of the known lectin pathway. In the following method for measuring the cleavage activity of deMASP-2 C4, mannan-coated plastic reservoirs were incubated for 37 minutes with diluted rat serum to activate the lectin pathway. Since the primary antibody used in this ELISA assay is only recognized In human C4, diluted rat serum was also supplemented with human C4 (1.0 μg / ml). The reservoirs were then washed and assayed while human C4b was immobilized in the reservoirs using standard ELISA methods. The amount of C4b generated in this assay is a measure of MASP-2 dependent C4 cleavage activity. Anti-MASP-2 Fab2 at the selected concentrations was tested in this assay for its ability to inhibit C4 cleavage.

Methods: Costar Medium Binding 96-well plates were incubated overnight at 5 ° C with diluted mannan in 50 mM decarbonate buffer, pH 9.5 at 1.0 μg / 50 μl / well. Each 3X washable reservoir with 200 μl of PBS. The reservoirs were then blocked with 100 μl / 1% bovine serum albumin reservoir in PBS and incubated for one hour at room temperature with gentle shaking. Each reservoir was washed 3X with 200 µl PBS. Anti-MASP-2 Fab2 samples were diluted at selected Ca ++ and Mg ++ concentrations containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl 2, 2.0 mM CaCl 2, 0.1% gelatin pH 7.4) at 5 ° C. 1.0 μg / ml human C4 (Quidel) was also included in these samples. 0.5% rat serum was added to the above samples at 5 ° C and 100 µl were transferred to each reservoir. The plates were covered and incubated for 30 min in a 37 ° C water bath to allow complement activation. The reaction was stopped by transferring the 37 ° C water bath plates to a recipient containing an ice-water mixture. Each reservoir was washed 5 χ 200 μΐ with PBS-Tween 20 (0.05% Tween 20 in PBS), then each reservoir was washed 2X with 200 μΐ PBS. 100 μl / 1: 700 dilution reservoir deanti-C4 biotin conjugated human chicken (Immunsystem AB, Uppsala, Sweden) were added in PBS containing 2.0 mg / ml bovine albumin (BSA) and incubated one hour at room temperature. soft comagitation. Each reservoir was washed 5 χ 200 μl PBS. 100μl / well of 0.1 μg / ml peroxidase-conjugated streptavidin (Pierce Chemical # 21126) was added in PBS containing 2.0 mg / ml of BSA and incubated for one hour at room temperature on a gentle co-shaker shaker. Each reservoir was washed 5 χ 200 μΐ with PBS. 100μΐ / TMB peroxidase substrate reservoir (Kirkegaard & PerryLaboratories) was added and incubated at room temperature for 16min. The peroxidase reaction was stopped by the addition of 100μΐ / 1.0 M H3PO4 reservoir and OD450 was measured.

3. Anti-mouse MASP-2 Fab2 Binding Assay to 'Native' mouse MASP-2

Background: MASP-2 is usually present in plasma as a dimeric MASP-2 complex which also includes specific lectin molecules (mannose binding protein (MBL) and phytolines). Therefore, if a person is interested in studying anti-Fab2 binding MASP-2 For the physiologically relevant form of MASP-2, it is important to develop a binding assay in which the interaction between Fab2 and 'native' MASP-2 in plasma is used, rather than purified recombinant MASP-2. The 10% serum 'native' MASP-2-MBL complex was first immobilized on mannan-coated reservoirs. The binding affinity of various anti-MASP-2 Fab2s to immobilized native MASP-2 was then studied using a standard ELISA methodology.

Methods: Costar High Binding 96-well plates were incubated overnight at 5 ° C with 50 mM diluted manganese carbonate buffer, pH 9.5 at 1 μ§ / 50 μΐ / well. Each 3X washable reservoir with 200 μΐ PBS. The reservoirs were blocked with 100μΐ / 0.5% skimmed milk powder reservoir in PBST (PBS with 0.05% Tween 20) and incubated for one hour at room temperature with gentle coitation. Each well was washed 3X with 200 μΐ TBS / Tween / Ca 2 Wash Buffer (Tris buffered saline, 0.05% Tween 20 containing 5.0 mM CaCl 2, pH 7.4). 10% rat serum in High Salt Binding Buffer (20 mM Tris, 1.0 M NaCl, 10 mM CaCl 2, 0.05% Triton-X 100, 0.1% (w / v) albumin bovine serum, pH 7.4) was prepared on ice. 100 μl / well was added and incubated overnight at 5 ° C. The wells were washed 3X with 200 µl TBS / Tween / Ca ++ Wash Buffer. The reservoirs were then washed 2X with 200 μl PBS. 100 μl / selected concentration reservoir of anti-MASP-2 Fab2 diluted in GVB Buffer containing Ca ++ eMg ++ (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl2, 2.0 mM CaCl2, 0.1% gelatin, pH 7.4) were added and incubated for one hour at room temperature with gentle shaking. Each reservoir was washed 5 χ200 μl PBS. 100 μl / well of HPR-conjugated goat anti-Fab2 (Biogenesis Cat No 0500-0099) diluted 1: 5000 in 2.0 mg / ml bovine serum albumin in PBS were added and incubated for one hour at room temperature with gentle shaking. Each reservoir was washed 5 χ 200 μl PBS. 100 μl / well of TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 70 min. The peroxidase reaction was stopped by the addition of 100 μl / 1.0 M H3PO4 reservoir and OD450 was measured.

Approximately 250 different Fab2s that reacted with high

affinity for rat MASP-2 protein were chosen for ELISA screening. These high affinity Fab2s were sequenced to determine the different antibody oddity and 50 unique anti-MASP-2 antibodies were purified for further analysis. 250 μg of each purified Fab2 antibody was used to characterize the binding affinity of deMASP-2 and to test the complement functional pathway. The results of this analysis are shown below in TABLE 6.Table 6: anti-MASP-2 FAB2 that blocks lectin pathway complement activation

<table> table see original document page 217 </column> </row> <table>

As shown above in TABLE 6, of the 50 anti-MASP-2 Fab2s tested, seventeen Fab2s were identified as MASP-2 blocker Fab2 that potently inhibit the formation of C3convertase with IC50 equal to or less than 10 nM Fab2s (a positive unclear rate). 34%). Eight of the seventeen identified Fab2s have IC50s in the subnanomolar range. In addition, all seventeen of the MASP-2 blocking Fab2s shown in TABLE 6 gave essentially complete inhibition of C3 convertase formation in the C3convertase lectin pathway assay. FIGURE 11A graphically illustrates the results of the C3 convertase deformation assay for Fab2 # 11 antibody, which is representative of the other Fab2 antibodies tested, the results of which are shown in TABLE 6. This is an important consideration as it is theoretically possible that a Fab2 "blocker" can only fractionally inhibit MASP-2 function even when each MASP-2 molecule is bound by Fab2.

Although mannan is a known lectin pathway activator, it is theoretically possible that the presence of rat serum anti-mananane antibodies may also activate the classical pathway and generate C3b via the classical pathway C3b convertase. However, each of the seventeen anti-MASP-2 blocking Fab2s listed in this example potently inhibit C3b generation (> 95%), thus demonstrating the specificity of this assay for lectin pathway C3 convertase.

Binding assays were also performed with all seventeen of the blocking Fab2s to calculate an apparent Kd for each. Results of native mouse MASP-2 anti-mouse FabP2 Fab2s binding assays for six of the blocking Fab2s are also shown in TABLE 6. FIGURE IIB graphically illustrates the results of a Fab2 # 11 antibody binding assay. Similar binding assays were also performed for the other Fab2s, the results of which are shown in TABLE 6. Overall, the apparent Kds obtained for targeting each of the six Fab2s to 'native' MASP-2 would correspond fairly well with the IC50 for Fab2 C3convertase functional assay. There is evidence that MASP-2 undergoes a conformational change from 'inactive' to 'active' in the activation of its protease activity (Feinberg et al., EMBO J 22: 2348-59 (2003); Gal et al, J Biol Chem 280: 33435-44 (2005)). In normal rat plasma used in the C3 convertase formation assay, MASP-2 is primarily present in the zymogen conformation. In contrast, in the binding assay, MASP-2 is present as part of a mannan-immobilized MBL-linked complex; therefore, MASP-2 may be in the 'active' conformation (Petersenet al, J. Immunol Methods 257: 107-16, 2001). Consequently, one would not necessarily expect an exact match between the IC50 and Kd for each of the seventeen Fab2 blockers tested in these two functional assays since in each assay Fab2 would be binding a different conformational form of MASP-2. However, with the exception of Fab2 # 88, there appears to be a reasonably close match between the IC50 and apparent Kd for each of the other sixteen Fab2 tested in both trials (see TABLE 6).

Several of the blocking Fab2s were evaluated for inhibition of C4 MASP-2 mediated cleavage. FIGURE Illustratively illustrates the results of a C4 cleavage assay showing inhibition with Fab2 # 41, with an IC 50 = 0.81 nM (see TABLE 6). As shown in FIGURE 12, all of the Fab2s tested were found to inhibit C4 binding with IC50s similar to those obtained in the C3convertase assay (see TABLE 6).

Although mannan is a known lectin pathway activator, it is theoretically possible that the presence of rat serum anti-mannan antibodies would also activate the classical pathway and thus generate C4b by C4 Cls mediated cleavage. However, several anti-MASP-2 Fab2s have been identified that potently inhibit C4b generation (> 95%), thus demonstrating the specificity of this assay for MASP-2 mediated C4 cleavage. C4, like C3, contains an unusually highly reactive thioester group as part of its structure. In cleavage of C4 by MASP-2 in this assay, the C4b thioester group can form a covalent bond with hydroxyl or amino groups to macromolecules immobilized at the bottom of the plastic reservoirs via ester or amide bonds, thereby facilitating the detection of C4b in the ELISA assay.

These studies clearly demonstrate the creation of high affinity FAB2s for rat MASP-2 protein that functionally block the activity of both C4 and C3 convertase, thereby preventing lectin pathway activation.

EXAMPLE 25

This example describes epitope mapping for various of the blocking anti-mouse MASP-2 Fab2 antibodies that were generated as described in Example 24.

Methods:

As shown in FIGURE 13, the following proteins, all with N-terminal 6X His labels were expressed in CHO cells using the pED4 vector:

Rat MASP-2A, a life-size, inactivated MASP-2 protein inactivating serine at the active center for alanine (S613A);

Rat MASP-2K, a life-size altered MASP-2 protein to reduce self-activation (R424K);

CUBI-II, a mouse MASP-2 N-terminal fragment containing only the CUBI domains, equivalent to EGF and CUBII; and

CUBI / EGF equivalent, a mouse deMASP-2 N-terminal fragment containing only the CUBI domains and EGF equivalent.

These proteins were purified from the culture supernatants by nickel affinity chromatography as previously described (Chen et al., J. Biol. Chem. 276: 25894-02 (2001)).

A C-terminal polypeptide (CCPII-SP), containing CCPII and the rat MASP-2 serine protease domain, was expressed in E. colic as a thioredoxin fusion protein using pTrxFus (Invitrogen). Aprotein was purified from cell lysates using Thiobond deafness resin. The thioredoxin fusion partner was expressed from empty pTrxFus as a negative control.

All recombinant proteins were dialyzed in TBS buffer and their concentrations determined by measuring OD at 280 nm. DOT BLOT ANALYSIS:

Serial dilutions of the five recombinant MASP-2 polypeptides described above and shown in FIGURE 13 (and thioredoxin polypeptide as a negative control for CCPII-serine protease polypeptide) were stained on a nitrocellulose membrane. Amount of stained protein ranged from 100 ng to 6.4 μ§ in five-fold steps. In later experiments, the amount of spotted protein ranged from 50 ng to below 16 pg, again in five-fold steps. Membranes were blocked with 5% skimmed milk powder in TBS (blocking buffer) after incubation with 1.0 μg / ml anti-MASP-2 Fab2s in blocking buffer (containing 5.0 mM Ca2 +). Bound Fab2s were detected using HRP-conjugated anti-human Fab (AbD / Serotec; diluted 1 / 10,000) and an ECL detection kit (Amersham). One membrane was incubated with anti-polyclonal rabbit human Ab Ab (described in Stover et al. al., J. Immunol 163: 6848-59 (1999)) as a positive control. In this case, bound Ab was detected using HRP-conjugated goat anti-rabbit IgG (Dako; diluted 1 / 2,000).

MASP-2 Binding Assay

ELISA plates were coated with 1.0 μg / recombinant MASP-2A reservoir or CUBI-II polypeptide in carbonate buffer (pH 9.0) overnight at 4 ° C. Reservoirs were blocked with 1% BSA in TBS, then serial dilutions of the anti-MASP-2 Fabs added in TBS containing 5.0 mM Ca2 +. The plates were incubated for one hour at room temperature. After washing three times with TBS / tween / Ca2 +, HRP-conjugated anti-human Fab (AbD / Serotec) diluted 1 / 10,000 in TBS / Ca was added and the plates incubated for an additional one hour at room temperature. Bound antibody was detected using a TMB peroxidase substrate kit (Biorad).

RESULTS:

Results of spot spot analysis demonstrating the reactivity of Fab2s with various MASP-2 polypeptides are given below in TABLE 7. The numerical values provided in TABLE 7 indicate the amount of spotted protein required to approximate the semi-maximal signal strength. As shown, all polypeptides (with the exception of the thioredoxin fusion partner alone) were recognized by the positive control Ab (polyclonal anti-MASP-2 human sera, incited in rabbits).

Table 7: Reactivity with various recombinant rat MASP-2 polypeptides at spot spots

<table> table see original document page 222 </column> </row> <table>

NR = No reaction. The positive control antibody is anti-HIV sera.

Polyclonal human MASP-2, incited in rabbits.

All Fab2s reacted with MASP-2A as well as MASP-2K (data not shown). Most Fab2s recognized the CCPII-SP polypeptide but not the N-terminal fragments. The two exceptions are Fab2 # 60 and Fab2 # 57. Fab2 # 60 recognizes MASP-2A and the CUBI-II fragment, but not the CUBI / EGF equivalent polypeptide or CCPII-SP polypeptide, suggesting that it binds to a CUBIT or transposing epitope and the EGF equivalent. Fab2 # 57 recognizes MASP-2A but not any of the MASP-2 fragments tested, perhaps indicating that this Fab2 recognizes an epitope on CCP1. Fab2 # 40 and # 49 only linked to the full MASP-2A. In the ELISA binding assay shown in FIGURE 14, Fab2 # 60 also bound to the CUBI-II polypeptide, although with slightly lower apparent affinity.

These findings demonstrate the identification of multiple blocking Fab2s to the multiple protein regions of MASP-2. EXAMPLE 26

This example describes the analysis of MASP-2 - / - mice in a Murine Renal Ischemia / Reperfusion Model.

Rationale / Rational analysis: Ischemia-Reperfusion (I / R) injury to the kidney at body temperature has relevance in several clinical conditions, including hypovolemic shock, renal artery occlusion, and cross-clamping procedures.

Renal ischemia-reperfusion (I / R) is a major cause of acute renal failure, associated with a mortality rate of up to 50% (Levy et al., JAMA 275: 1489-94, 1996; Thadhani et al., N. Engl. J. Med. 334: 1448-60, 1996). Renal failure after transplantation is a common and life threatening complication after kidney transplantation (Nicholson et al., Kidney Int. 58: 2585-91, 2000). Effective treatment for renal I / R alleviation is not currently available and hemodialysis is the only available treatment. The pathophysiology of renal I / R injury is complicated. Recent studies have shown that the lectin pathway of complement activation may play an important role in the pathogenesis of renal I / R injury (deVries et al., Am. J. Path. 165: 1677-88, 2004). Methods:

A MASP-2 (- / -) mouse was generated as described in Example 1 and backcrossed for at least 10 generations with C57B1 / 6. Six male MASP-2 (- / -) and six wild type (+ / +) mice weighing between 22 and 25 g were administered with an intraperitoneal injection of Hypnovel (6.64 mg / kg; Roche products Ltd. Welwyn Garden City, UK) subsequently anesthetized by isoflurane inhalation (AbbottLaboratories Ltd., Kent, UK). Isoflurane was chosen because it is a mild inhalation anesthetic with minimal liver toxicity; concentrations are accurately produced and the animal recovers rapidly even after prolonged anesthesia. Hypnovel was administered because it produces a neuroleptanalgesia condition in the animal and means that lessisoflurane needs to be administered. A cushion that has been placed behind the animal to maintain a constant body temperature. Then a central abdominal incision was made and the body cavity kept open using a pair of retractors. The connective tissue was removed above and below the renal vein and artery from both the right and left kidney, and the renal pedicle was clamped by the application of demicroaneurysm clamps for a period of 55 minutes. This period of ischemia was initially based on an earlier study conducted in this laboratory (Zhou et al., J. Clin. Invest. 105: 1363-71 (2000)). In addition, a standard 55-minute ischemic time was chosen following ischemic titration, and 55 minutes was found to give compatible lesion that was also reversible, with low mortality of less than 5%. After occlusion, 0.4 ml of warm (37 ° C) saline solution was placed in the abdominal cavity and then the abdomen was closed during the ischemia period. Following removal of the microaneurysm staples, the kidneys were observed until color change, an indication of blood re-flow to the kidneys. An additional 0.4ml of warm saline solution was placed in the abdominal cavity and the opening was sutured, after which the animals were returned to their cages. Tail blood samples were taken 24 hours after staple removal and 48 hours after the mice were removed. were sacrificed and an additional blood sample was collected.

Renal Injury Assessment: Renal function was assessed 24 and 48 hours after reperfusion in six male MASP-2 (- / -) and WT (+ / +) mice. Blood creatinine measurement was determined by mass spectrometry, which provides a reproducible index of renal function (sensitivity <1.0 μπιοΙ / L). FIGURE 15 graphically illustrates urea nitrogen clearance for wild-type eMASP-2 (- / -) C57B1 / 6 controls at 24 hours and 48 hours after reperfusion. As shown in FIGURE 15, MASP-2 (- / -) mice demonstrated a significant reduction in the amount of blood urea at 24 and 48 hours compared to wild type control mice, indicating a protective functional effect of kidney injury in the model. ischemia-reperfusion injury.

From end to end, increased blood urea was observed in both WT (+ / +) and MASP-2 (- / -) mice at 24 and 48 hours following surgical procedure and ischemic insult. Blood urea levels in a non-ischemic WT (+ / +) animal surgery were separately determined to be 5.8 mmol / L. In addition to the data presented in FIGURE 15, a MASP-2 (- / -) animal showed almost completed protection ischemic insult, with values of 6.8 and 9.6 mmol / L at 24 and 48 hours, respectively. This animal was excluded from group analysis as a potential testimony, in which no ischemic injury could have been present. Therefore, the final analysis shown in FIGURE 15 included 5 MASP-2 (- / -) mice and 6 WT (+ / +) mice and a statistically significant reduction in blood urea was observed at 24 and 48 hours in MASP-2 (- /) mice. -) (Student's t test ρ <0.05). These findings indicate that inhibition of MASP-2 activity would be expected to have a protective or therapeutic effect of renal injury due to ischemic injury.

Example 27

This example describes the analysis of MASP-2 (- / -) mice.

in a Mouse Myocardial Ischemia / Reperfusion Model.

Rationale / Rational Analysis:

Mannose-binding lectin (MBL) is a circulating molecule that initiates complement activation in an immune-independent complex manner in response to a wide range of carbohydrate structures. These structures may be components of infectious agents or altered endogenous carbohydrate moieties particularly within necrotic, oncotic or apoptotic cells. These forms of cell death occur in reperfused nomocardium where complement activation probably expands the lesion beyond the boundary that exists at the time when ischemia is terminated by reperfusion. Although there is convincing evidence that complement activation aggravates myocardial reperfusion, the mechanism of such activation is not well understood and inhibition of all known pathways is likely to have intolerable adverse effects. A recent study suggests that activation may involve MBL rather than the classical path or alternative amplification loop (as defined in the present invention), since infarction was reduced in MBL-null (AZC) mice but not in Clq-null (Walsh) mice. MC et al., Jour of Immunol 175: 541-546 (2005)) However, while encouraging, these mice still harbor circulating components, such as Ficoline A, capable of activating complement via the lectin pathway.

This study investigated MASP-2 (- / -) versus wild type (+ / +) controls to determine if MASP-2 (- / -) would be less sensitive to myocardial ischemia and reperfusion injury. MASP-2 (- / -) mice underwent regional ischemia and the infarct size was compared with their wild-type littermates.

Methods: The following protocol was based on a procedure to induce ischemia / reperfusion injury previously described by Marber et al., J. Clin Invest. 95: 1446-1456 (1995)).

A MASP-2 (- / -) mouse was generated as described in Example 1 and backcrossed for at least 10 generations with C57B1 / 6. Seven MASP-2 (- / -) and seven (+ / +) wild-type mice were anesthetized with ketamine / medetomidine (100 mg / kg and 0.2 mg / kg, respectively) and placed on their backs on a thermostatically warming pad. controlled to maintain the rectal temperature at 37 ± 0.3 ° C. The mice were intubated under direct vision and ventilated with ambient air at a respiration rate of 11 O / min and a tidal volume of 250 O / min (Ventilator - Hugo Sachs Elektronic MiniVent Type 845, Germany).

The hair was shaved and an incision of anterolateral skin made from the left axilla to the processus xiphoideus. The pectoralismajor muscle was dissected, cut at its outer margin, and moved in the axillary pit.

The pectoralis minor muscle was cut at its ecaudally moved cranial margin. The muscle was later used as an abamuscular covering the heart during coronary artery occlusion. The muscles of the 5th intercostal space and the pleura parietalis were penetrated into a slightly mediated point in relation to the left lung margin, thus avoiding injury to the lung or heart. After pleura penetration the forceps were carefully directed beyond the pleura towards the sternum without touching the heart and the pleura and the intercostal muscles were dissected with a battery-powered cauterizer (Harvard Apparatus, UK). Special care has been exercised in avoiding any bleeding. Using the same technique, the thoracotomy was extended to the soft axillary line. After cutting the 4th rib on its outer edge of the intercostal space was enlarged until the whole heart was exposed from the base to the apex.

With two small artery forceps, the pericardium was opened and a talapericardial was made to move the slightly anterior heart. The left anterior descending coronary artery (LAD) was exposed and an 8-0 demonofilament suture with a curved needle was then passed under the LAD. The LAD-binding site resides exactly caudally from the tip of the left atrium, about 1/4 of the length of the line going from the atrioventricular crest to the left ventricular apex.

All experiments were performed in a blind manner, with the investigator not being aware of the genotype of each animal. After completion of the instrumentation and surgical procedures, the mice were left in a 15 min equilibration period. The mice then underwent 30 min of coronary artery occlusion with 120 min of reperfusion time.

Coronary artery occlusion and reperfusion model

Coronary artery occlusion was obtained using the hanging weight system as previously described (Eckle et al., Am J PhysiolHeart Circ Physiol 29 /: H2533-H2540, 2006). Both ends of the monofilament cuff were passed through a 2 mm long piece of a PE-10 polythene tube and attached to a 5-0 suture length using cyanoacrylate glue. The suture was then directed to two horizontally mounted mobile demetal rods and masses of 1 g each were attached to both ends of the suture. By lifting the stems, the masses were suspended and the suture placed under controlled tension to occlude the LAD with a defined and constant pressure. The occlusion of the LAD was verified by the pale of the area at risk, changing the color of the LAD perfusion zone from living red to violet, indicating the cessation of blood flow. The perfusion was obtained by lowering the rods until the masses rested on the operating pads and the ligature tension was relieved. Perfusion was verified by the same three criteria used to verify occlusion. Mice were excluded from further analysis if all three criteria were not met at the beginning of coronary artery occlusion or within 15 min of reperfusion, respectively. During coronary artery occlusion, the temperature and humidity of the heart surface were maintained by covering the heart with the pectoralis minor muscle flap and sealing the thoracotomy with a 0.9% saline wet gauze. of the myocardium:

Infarction size (ESiF) and area at risk (AAR) were determined by planometry. After i.v. injection of 500 I.U. of heparin LAD was re-occluded and 300 μΐ 5% Evans Blue (w / vol) (Sigma-Aldrich, Poole, UK) were slowly injected into the jugular vein to delineate the area at risk (AAR). This causes the dye to enter the nonischemic region of the left ventricle and leave the dyed ischemic RAA. After the mice were euthanized by cervical dislocation, the heart was quickly removed. The heart was chilled on ice and mounted on a 5% agarose block and then cut into 8 transverse slices of 800 μηι thickness. All slices were incubated at 37 ° C for 20 min with 3% 2,3,5-triphenyltetrazolium chloride (Sigma Aldrich, Poole, UK) dissolved in pH 7.4 adjusted 0.1 M Na2HPO4ZNaH2PO4 buffer. The slices were fixed overnight in 10% formaldehyde. The slices were placed between two coverslips and the sides of each slice were digitally imaged using a high resolution optical scanner. Digital images were then analyzed using SigmaScan software (SPSS, US). The size of the infarcted area (light), the left ventricular (LV) area, reddening (red), and the normally perfused LV zone (blue) were outlined in each section by identifying its color appearance and colored borders. The areas were quantified in both the sides of each slice are averaged by an investigator. Infarct size was calculated as a% of the risk zone for each animal.

RESULTS: The size of the infarcted area (light), the endangered LV area (red), and the normally perfused LV zone (blue) were outlined in each section by identifying their color appearance and colored edges. The areas were quantified on both sides of each slice and averaged by an investigator. Infarct size was calculated as a% of the risk zone for each animal. FIGURE 16A shows the evaluation of seven WT (+ / +) and seven MASP-2 (- / -) mice for determination of their infarct size after undergoing the coronary artery occlusion and reperfusion technique described above. As shown in FIGURE 16A, MASP-2 (- / -) mice demonstrated a statistically significant (p <0.05) reduction in infarct size versus wild type (+ / +) mice, indicating a myocardial injury-protective effect in the injury model. of ischemia-reperfusion. FIGURE 16B shows the distribution of individual animals tested, indicating a clear protective effect for MASP-2 (- / -) mice.

EXAMPLE 28

This example describes the results of MASP-2 - / - in a Murine Macular Degeneration Model.

Rationale / Rational analysis: Age-related macular degeneration (AMD) is the leading cause of blindness after age 55 in the industrialized world. AMD occurs in two main forms: neovascular AMD (wet) and atrophic AMD (dry). The neovascular (wet) form accounts for 90% of the severe visual loss associated with AMD, although only -20% of individuals with AMD develop the wet form.

AMD's clinical hallmarks include multiple colloid bodies, atrophygeography, and choroidal neovascularization (CNV). In December 2004, the FDA approved Macugen (pegaptanib), a new class of ophthalmic drugs to specifically target and block the effects of vascular endothelial decay factor (VEGF), for the treatment of the wet (neovascular) form of AMD (Ng et al, Nat Rev. Drug Discov 5: 123-32 (2006)).

Although Macugen represents a promising new therapeutic option for a subset of AMD patients, there remains a pressing need to develop additional treatments for this complex disease. Research-independent multiple lines play a central role in complement activation in the pathogenesis of AMD. The pathogenesis of choroidal neovascularization (CNV), the most serious form of AMD, may involve activation of complement pathways.

More than twenty-five years ago, Ryan described a model of CNV laser-induced injury in animals (Ryan, S. J., Tr. Am. Opth.

Sound. LXXVII: 707-745, 1979). The model was initially developed using rhesus monkeys, however, the same technology has since been used to develop similar CNV models in a variety of animal research, including the mouse (Tobe et al., Am. J. Pathol.153: 1641-46 , 1998). In this model, laser photocoagulation is used to break the Bruch membrane, an action that results in CNV-equivalent membrane formation. The salt-induced model captures many important traits of the human condition (for a recent review, see Ambati et al., Survey Ophthalmology 48: 257-293, 2003). The laser-induced mouse model is now well established and is used as an experimental basis in a large and ever-growing number of research projects. It is generally accepted that the laser-induced model shares sufficient biological similarity with CNV in humans that clinical studies of pathogenesis and drug inhibition using this model are relevant to CNV in humans.

Methods:

A MASP-2 - / - mouse was generated as described in Example 1 and backcrossed for 10 generations with C57B1 / 6. The current study compared the results when male MASP-2 (- / -) and MASP-2 (+ / +) mice were evaluated in the course of laser-induced CNV, an accelerated neovascular AMD model focused on laser-induced CNV volume by confocal microscopy. scanning laser as a measure of tissue injury and determination of VEGF levels, a potent angiogenic factor implicated in CNV, in retinal pigment epithelium (RPE) / choroid by ELISA after laser injury. Choroidal neovascularization (CNV) induction: A

Laser photocoagulation (532 nm, 200 mW, 100 ms, 75Am; Oculeve GL, Iridex, Mountain View, CA) was performed on both eyes of each animal on days zero by a single individual masked to the designated drug group. The laser spots were applied in a standard manner around the optic nerve, using a slit-release system and a blade as a contact lens. The morphological end point of the laser lesion was the appearance of a cavitation bubble, a sign considered to correlate with the rupture of the Bruch membrane. The detailed methods and endpoints that were evaluated are as follows.

Fluorescein Angiography: Fluorescein angiography was performed with a camera and imaging system (TRC 50 IA camera; ImageNet 2.01 system; Topcon, Paramus, NJ) at week 1 after laser photocoagulation. Photographs were captured with a 20-D lens in contact with the background camera lens after intraperitoneal injection of 0.1 ml 2.5% sodium fluorescein. A retinan expert not involved in laser photocoagulation or angiography evaluated the fluorescein angiograms in a single session in the masked manner.

Choroidal neovascularization volume (CNV): One week after the laser lesion, the eyes were enucleated and fixed with 4% paraformaldehyde for 30 min at 4 ° C. The eyeglasses were obtained by removing the anterior segments and were washed three times in PB S, followed by dehydration and rehydration through a series of methanol. After blocking twice with buffer (PBS containing 1% bovine albumin and 0.5% X-100) for 30 minutes at room temperature, eye glasses were incubated overnight at 4 ° C with 0.5% FITC-isolectin B4 (Vector laboratories, Burlingame, CA), diluted with 0.2% PBS of BSA and 0.1% Triton X-100, which binds terminal β-D-galactose residues on endothelial cell surfaces and selectively labels the murine vasculature. After two washes with PBS containing 0.1% Triton X-100, the sensorine retina was gently detached and cut from the optic nerve. Four relaxing radial incisions were made and the rest of the RPE — remnant choroid-sclerotic complex was mounted flat on an anti-wilt medium (Immu-Mount Vectashield Mounting Medium; Vector Laboratories) and covered with a coverslip.

The flat mounts were examined with a laser scanning focal microscopy (TCS SP; Leica, Heidelberg, Germany). Bones were visualized by excitation with the blue wavelength (488 nm) and capture emission between 515 and 545 nm. A 40X oil immersion object was used for all imaging studies. The horizontal optical sections (1 μηι pitch) were obtained from the surface of the RPE-choroid-sclerotic complex. The deepest focal plane in which the surrounding choroidal vascular network connecting the lesion could be identified was deemed to be the floor of the lesion. Any vessel in the laser-targeted area superficial to this reference plane was judged as CNV. The images from each section were digitally stored. CNV-related fluorescence area was measured by computerized image analysis using the microscope software (TCS SP; Leica). The sum of the entire fluorescent area in each horizontal section was used as an index for CNV volume. Imaging was performed by an operator masked by the treatment group designation.

Because the likelihood of each laser lesion developing CNV is influenced by the group to which it belongs (mouse, eye and laser spot), the average lesion volumes were compared using a linear mixed model with a repeated-measured slot-depletion planning. The entire plotting factor was the genetic group to which the animal belongs, while the slit plotting factor was the eye. Statistical significance was determined at the 0.05 level. Posthoc mean comparisons were constructed with a Bonferroni adjustment for multiple comparisons.

VEGF ELISA. Three days after the 12-spot laser injury, the RPE-choroid complex was sonicated in Iise buffer (20mM deimidazole HCl, 10mM KCl, 1mM MgCl2, 10mM EGTA, 1% Tritron X-100, 10 mM NaF, 1 mM Na molybdate and 1 mM EDTA with protease inhibitor) on ice for 15 min. Supernatant VEGF protein levels were determined by an ELISA kit (R&D Systems, Minneapolis, MN) that recognizes all junction variants, 450 to 570nm (Emax; Molecular Devices, Sunnyvale, CA) and normalized to total protein. Duplicate measurements were performed in a masked manner by an operator not involved in photocoagulation, image formation or angiography. VEGF numbers were plotted as the mean +/- SEM of at least three independent experiments and compared using the Mann-Whitney U test. The null hypothesis was rejected at P <0.05.

RESULTS:

Assessment of VEGF Levels:

FIGURE 17A graphically illustrates VEGF protein levels in the isolated RPE-choroid complex of C57B16 wild type and MASP-2 (- / -) mice on day zero. As shown in FIGURE 17A, assessment of VEGF levels indicates a decrease in VEGF reference levels in MASP-2 (- / -) mice versus wild-type C57bl control mice. FIGURE 17B graphically illustrates the VEGF protein levels measured over the three days following laser-induced injury. As shown in FIGURE 17B VEGF levels were significantly increased in wild type (+ / +) mice three days following laser-induced injury, consistent with published studies (Nozaki et al., Proc. Natl. Acad. Sci. USA 103: 2328-33 (2006)). However, surprisingly very low levels of VEGF have been observed in MASP-2 (- / -) mice Choroidal neovascularization (CNV) evaluation:

In addition to the reduction in VEGF levels following laser-induced muscle degeneration, the area of CNV was determined before and after laser damage. FIGURE 18 graphically illustrates the volume of CNV measured in wild type C57bl mice and MASP-2 (- / -) day seven mice following laser-induced injury. As shown in FIGURE 18, MASP-2 (- / -) mice demonstrated a reduction of about 30% in the area of CNV following laser-induced injury on day seven compared to wild type control mice.

These findings indicate a reduction in VEGF and CNV as seen in MASP (- / -) mice versus wild type (+ / +) control and that blocking MASP-2 with an inhibitor would have a preventive or therapeutic effect in the treatment of macular degeneration.

EXAMPLE 29

This example describes the results of MASP-2 (- / -) in a Murine Monoclonal Antibody-Induced Rheumatoid Arthritis Model

Rationale / Rational analysis: The most animal model

Commonly used for rheumatoid arthritis (RA) is collagen-induced arthritis (CIA) (for recent reviews, see Linton and Morgan, Mol. Immunol.36: 905-14, 1999). Type II collagen (IIC) is a major constituent of joint matrix proteins and immunization with adjuvant native IIC induces autoimmune polyarthritis by a cross-reactive autoimmune response to IIC in the joint cartilage. As in RA, susceptibility to CIA is linked to expression of a certain class of II MHC alleles. Some mouse strains, including the C57B1 / 6 strain, are resistant to classical ASD because they lack an appropriate MHC haplotype and therefore do not generate high anti-CII antibody titers. However, it has been found that compatible arthritis can be induced in all strains of mice by i.v. or i.p. in mice from a cocktail of four specific monoclonal antibodies against type II collagen. These arthidogenic monoclonal antibodies are commercially available (Chondrex, Inc., Redmond, WA). This passive CIA transfer model has been used with textiles in several recent published reports using the C57B1 / 6 mouse strain (Kagari et al., J. Immunol. 169: 1459-66, 2002; Kato et al., J. Rheumatol. 30: 247 -55, 2003; Banda et al, J. Immunol. 177: 1904-12, 2006).

The following study compared the sensitivity of wild type (WT) and MASP-2 (- / -) mice, both sharing the genetic foundations of C57B1 / 6, for the development of arthritis using the passive CIA transfer model.

Methods:

Animals: A MASP-2 (- / -) mouse was generated as described in Example 1 and backcrossed for 10 generations with C57B1 / 6. Fourteen male and female wild-type C57BL / 6 mice were seven to eight weeks old at the time of edez antibody injection C57B1 / 6 MASP-2 (- / -) and wild (+ / +) male and female mice that were seven to eight weeks old at the time of antibody injection were used in this study. Twenty mice were injected with a monoclonal antibody cocktail to obtain 20 solid responders (two groups of ten). The animals (ten / group) were housed with five animals / cage and acclimatized for five to seven days before starting the study.

Mice were injected intravenously with a monoclonal antibody cocktail (Chondrex, Redmond WA) (5 mg) on day 0 and day 1. The test agent was a Chondrex + LPS monoclonal antibody. On day 2, the mice were dosed ip with LPS. . The mice were weighed on days 0, 2, 4, 6, 8, 10, 12 and before completion on day 14. On day 14 the mice were anesthetized with isoflurane and terminally bled for serum. After blood collection, the mice were euthanized, with both hind and forelimbs removed with the knees, which were placed in formalin for further processing.

Treatment Groups:

Group 1 (control): 4 mice of strain C57 / BL / 6 WT (+ / +);

Group 2 (test): 10 mice of strain C57 / BL / 6 WT (+ / +) (received cocktail mAb plus LPS); and

Group 3 (test): 10 mice of strain C57 / BL / MASP-2K0 / 6AÍ (- / -) (received cocktail mAb plus LPS)

Clinical arthritic counts were evaluated daily using the following counting system: 0 = normal; 1 = 1 affected front or rear joint; 2 = 2 affected front or rear foot joints; 3 = 3 affected front or rear foot joints; 4 = moderate (moderate erythema and swelling or 4 affected finger joints); 5 = severe (patella with diffuse erythema and severe swelling, unable to flex the fingers)

Results:

FIGURE 19 shows the group data plotted for average daily clinical arthritis event for up to two weeks. No clinical arthritis counts were observed in the control group that did not receive CoL2 MoAb treatment. MASP (- / -) mice had a lower clinical arthritis count from day 9 to day 14. Overall clinical arthritis count with area under curve analysis (AUC) indicated a 21% reduction in the MASP-2 group (- / -) versus WT mice (+ / +). However, the C57B16 mouse fundamentals as discussed above do not provide a robust global clinical arthritis count.

Due to the small incidence rate and group size, although tendentially, the data provided only tended (p = 0.1) and were not statistically significant at the ρ level <0.05. Additional animals in the treatment groups would be needed to show statistical significance. Due to the reduced incidence of arthritis, footpad counts were evaluated for severity. No single incidence of a clinical arthritis count of more than 3 was observed in any of the MASP-2 (- / -) mice, which was observed in 30% of WT (+ / +) mice, further suggesting that (1) the severity arthritis may be related to complement pathway activation and (2) that MASP-2 obliqueness may have a beneficial effect on arthritis.

Example 30

This example demonstrates that the Small Lectin Binding Mannose Associated Protein (Mapl9 or sMAP) is a MASP-2 dependent complement activation inhibitor.

Rationale / Rational Analysis:

Abstract:

Mannose-binding lectin (MBL) and phytolins are standard recognition proteins that act on innate immunity and trigger activation of the lectin complement pathway through MBL-serine-associated proteases (MASPs). On activation of the lectin pathway, MASP-2 cleaves C4 and C2. MBL-associated small protein (sMAP), a truncated form of MASP-2, is also associated with MBL / phycoline-MASP complexes. To clarify the role of sMAP, we generated sMAP-deficient mice (sMAP - / -) by targeted disruption of sMAP-specific exon. Because of gene disruption, the level of MASP-2 expression was also decreased in sMAP - / - mice when recombinant sMAP (rsMAP) and recombinant MASP-2 (rMASP-2) reconstituted the MBL-MASP-sMAP complex in serum. deficient, the binding of these recombinant MBLs was competitive and the C4 cleavage activity of the MBL-MASP-sMAP complex was restored by the addition of rMASP-2, while the addition of rsMAP attenuated the activity. Therefore, MASP-2 is essential for C4 activation and sMAP plays a regulatory role in lectin pathway activation.

Introduction:

The complement system mediates a protein complex chain reaction and assembly of protein complexes, plays a major role in biodefense as a part of both innate and adaptive immune systems. The mammalian complement system consists of three activation pathways, the classical pathway, the alternative pathway, and the lectin pathway (Fujita, Nat. Rev. Immunol. 2: 346-353 (2002); Walport, N Engl JMed 344: 1058- 1066 (2001)). The lectin pathway provides the primary lineage of defense against invading pathogens. The pathogen-recognizing components of this pathway, mannose-binding lectin (MBL) and phytolines, bind to the carbohydrate series on the surfaces of bacteria, viruses, and parasites and activate MBL-associated serum proteases (MASPs) to trigger a cascade. downstream reaction. The importance of the dalectin pathway for innate immune defense is underlined by several clinical studies linking an MBL deficiency with increased susceptibility to a variety of infectious diseases, particularly in early childhood before the adaptive immune system is established (Jack et al, Immunol Rev180: 86- 99 (2001); Neth et al., Infect Immun 68: 688-693 (2000); Summerfield et al., Lancet 345: 886-889 (1995); Super et al., Lancet 2: 1236-1239 (1989)) . However, the lectin pathway also contributes to unwanted complement activation, which is involved in tissue inflammation and inflammation in a variety of pathological conditions, including porischemia / perfusion injury to the heart and kidneys (de Vries et al., Am J Pathol 165: 1677- 1688 (2004); Fiane et al., Circulation 108: 849-856 (2003); Jordan et al., Circulation 104: 1413-1418 (2001); Walsh et al., J Immunol 175: 541-546 (2005)) .

As mentioned above, the lectin pathway involves carbohydrate recognition by MBL and phycolines (Fujita et al, Immunol Rev 198: 185-202 (2004); Holmskov et al, Annu Rev Immunol 21: 547-578 (2003); Matsushita and Fujita , Immunobiology 205: 490-497 (2002) and these electins form complexes with MASP-I (Matsushita and Fujita, J Exp Med176: 1497-1502 (1992); Sato et al, Int Immunol 6: 665-669 (1994); Takada et al, Biochem Biophys Res Commun 196: 1003-1009 (1993), MASP-2 (Thielet al, Nature 386: 506-510 (1997), MASP-3 (Dahl et al, Immunity 15: 127-135 (2001) and a truncated MASP-2 protein (MBL-associated small protein; sMAP or MApl9) (Stover et al, J Immunol 162: 3481-3490 (1999); Takahashi et al, Int Immunol 11: 8590863 (1999). Family members deMASP consist of six domains, two C1r / C1s / Uegf / bone morphogenetic protein (CUB) domains, one epidermal growth factor (EGF) equivalent domain, two complement control proteins (CCP), or repeating domains. short consensus statements (SCR) and a serinaprotease domain (Matsushita et al, Curr Opin Immunol 10: 29-35 (1998). MASP-2 esMAP are generated by the alternate junction from a single sMAP structural gene and consists of the first CUB domain (CUB 1), the EGF equivalent domain and an extra 4 amino acid at the C-terminal end encoded by a sMAP-specific exon. MASP-I and MASP-3 are also generated from a single gene by the alternative junction (Schwaeble et al, Immunobiology 205: 455-466 (2002). When MBL and phytolines bind to carbohydrates on the surface of microbes, the pro-enzyme form of MASP is cleaved between the second CCP and the protease domain, resulting in the formation consisting of two polypeptides, called heavy (H) and light (H) chains, thus acquiring proteolytic activities against breakdown components.Accumulated evidence shows that MASP-2 cleaves C4 and C2 (Matsushita et al, J. Immunol 165: 2637-2642 (2000) leading to the formation of C3 convertase (C4bC2a). We have proposed that MASP-I cleaves C3 directly and subsequently activates the amplification loop (Matsushita and Fujita). , Immunobiology 194: 443-448 (1995), but this function is controversial (Ambruset al, J. Immunol 170: 1374-1382 (2003). Although MASP-3 also contains an L-chain serine protease domain and exhibits its activity proteolytic against a sub synthetic substrate (Zundel et al, J Immunol 172: 4342-4350 (2004), their physiological substrates were not identified. The desMAP function lacking the serine protease domain remains unknown.

In the present study, to clarify the role of sMAP in activating the lectin complement pathway, we disrupted the sMAP exonespecific encoding 4 amino acid residues (EQSL) on the sMAP C-terminal end and generated sMAP - / - mice. We report here for the first time the ability of sMAP to downregulate lectin pathway activation.

Materials and methods

Mice

A targeting vector was constructed containing exons 1 to 4 and part of exon 6 of mouse 129 / Sv gene MASP-2 and a dogomy neomycin resistance cassette instead of exon 5 (Figure 20A). A DT-A gene was inserted at the 3 'end of the vector and three Iox ρ sites were inserted to perform conditional targeting to remove the deneomycin cassette and promoter region in the future. The targeting vector was subjected to electroporation in 129 / Sv. Targeted Es clones were microinjected into C57BL / 6J blastocysts that were implanted in the jutter of adoptive ICR mothers. Chimeric male mice formalcased with female C57BL / 6J mice to produce heterozygous (+/-) mice. (+/-) Heterozygous mice were screened by Southern blot analysis of BamHI-digested tail DNA using the probe indicated in FIGURE 20A. Southern blot analysis showed 6.5 kbp and 11 kbp bands in DNA from (+/-) heterozygous mice (Figure 20B). (+/-) heterozygous mice were backcrossed with C57BL / 6J mice. To obtain homozygous (- / -) mice, heterozygous (+/-) mice were intercrossed. Homozygous (- / -) mice (C57BL / 6J fundamentals) were identified by PCR-based genotyping of tail DNA. PCR analysis was performed using a mixture of exon 4 -specific and neo-gene-specific sense primers and an exon-6 antisense primer. DNA from (- / -) homozygous mice produced a single strand of 1.8 kbp (Figure 20C). In all experiments, 8 to 12 week old mice were used according to Fukushima Medical University guidelines for animal experimentation. Northern blot analysis

Poly (A) + RNA (1 μg) of wild type and homozygous (+ / +) mouse livers (- / -) was separated by electrophoresis, transferred to a nylon membrane and hybridized with a sPAP-specific 32P labeled cDNA probe , MASP-2 H chain, MASP-2 L chain or the neo gene. The same membrane was stripped and re-hybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specific probe.

Real-time PCR was performed with the LeveCycler System (Roche Diagnostics). Synthesized 60 ng cDNAs from wild type and homozygous (- / -) mouse deflated poly (A) + RNA (- / -) cDNAs were used as standard for real-time PCR and decade-old MASP H and L cDNA fragments -2 and sMAP were amplified and monitored.

The sample was electrophoresed on 10 or 12% SDS-polyacrylamide gels under reducing conditions and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Membrane proteins were detected with anti-MASP-1 antiserum against the MASP-I L chain or with anti-MASP-2 / sMAP antiserum against the MASP-2 H chain peptide.

MASPs and sMAP detection in MBL-MASPsAdAP complex

Mouse serum (20 μΐ) was added at 480 μΐ TBS-Ca2 + buffer (20 mM Tris-HCl, pH 7.4, 0.15 M NaCl and 5 mM CaCl2) containing 0.1% (w / v) BSA (TBS-Ca2 + / BSA) and incubated with 40 μΐ of 50% agarose manana gel paste (Sigma-Aldrich, St. Louis, MO) in TBS-Ca / BSA buffer at 4 ° C for 30 min. After incubation each gel was washed with TBS-Ca2 + buffer and SDS-PAGE sampling buffer was added to the gel. The gel was boiled and the supernatant was subjected to SDS-PAGE, followed by immunoblotting to detect MASP-1, MASP-2 and sMAP in the MBL complex.

C4 Deposition Test

Mouse serum was diluted with TBS-

Ca2 + / BSA up to 100 μΐ. The diluted sample was added to the mannan coated microtiter wells and incubated at room temperature for 30min. The wells were washed with ice-cold wash buffer (TBS-Ca2 + buffer containing 0.05% (v / v) Tween 20). After washing, human C4 was added to each reservoir and incubated on ice for 30min. The wells were washed with the cold wash buffer and HRP-conjugated anti-C4 human polyclonal antibody (Biogenesis, Poole, England) was added to each well. Following incubation at 37 ° C for 30 min, the reservoirs were washed with 3,3 ', 5,5'-tetramethylbenzidine (TMB) solution wash buffer and added to each reservoir. After development, 1 M H3PO4 was added and absorbance was measured at 450 nm.

C3 Deposition Test

Mouse serum was diluted with BBS buffer (4 mM debarbital, 145 mM NaCl, 2 mM CaCl 2 and 1 mM MgCl 2, pH 7.4) containing 0.1% (w / v) HSA to 100 μΐ. The diluted sample was added to the mannan coated microtiter reservoirs and incubated at 37 ° C for 1 hour. The reservoirs were washed with the wash buffer. After washing, HRP-conjugated anti-human C3c polyclonal antibody (Dako, Glostrup, Denmark) was added to each reservoir. Following incubation at room temperature for 1h, the reservoirs were washed with the wash buffer and TMB solution was added to each chamber. The color was measured as described above.

Recombinant

Recombinant sMAP mouse (rsMAP), rMASP-2, and inactive mutant MASP-2 mouse (MASP-2I) whose serine protease domain active desodium residue was replaced in place of alanine residue were prepared as described previously (Iwaki and Fujita , 2005).

MBL-MASP-sMAP complex reconstitution

Homozygous (- / -) mouse serum (20 μΐ) and various quantities of MASP-2I and / or rsMAP were incubated in a total volume of 40 μl in TBS-Ca2 + buffer on ice overnight. The mixture was incubated with agarose mannan-gel slurry and MASP-23 and rsMAP in the gel-bound MBL-MASP complex were detected as described in "Detection of MASPs and sMAP in MBL-MASP-sMAP Complex". C4 deposition

Homozygous (- / -) mouse serum (0.5 μl) and various amounts of rMASP-2 and / or rsMAP were incubated in a total volume of 20 μl in TBS-Ca2 + on ice overnight. The mixture was diluted with 80μl TBS-Ca2 + / BSA buffer and added to the mannan coated reservoirs. All subsequent procedures were performed as described in "C4 deposition assay".

FIGURE 20: The targeted disruption of the sMAP gene. (A) Partial restriction maps of the MASP-2 / sMAP gene, the targeting vector and the targeted allele. The sMAP-specific exon (exon 5) was replaced with a neo gene cassette. (B) Southern blot analysis of genomic DNA of progeny derived from mating chimeric male mice with female C57BL / 6J mice. The tail DNA was digested with BamHI and hybridized to the probe described in (A). An 11 kbp band was derived from the wild-type allele and a 6.5 kbp band from the targeted allele. (C) PCR genotyping analysis. The tail DNA was analyzed using a mixture of exon 4 -specific and genene-specific sense primers and an exon-6 antisense primer. A 2.5 kbp strip was obtained from the wild-type allele, a strip of 1.8 kb of the allele bleached.

FIGURE 21: Expression of sMAP and MASP-2 mRNAs in homozygous (- / -) mice. (A) Northern blot analysis. Poly (A) + wild-type and homozygous (- / -) mouse liver RNAs (- / -) were electrophoresed, transferred to a nylon membrane hybridized with a 32P-labeled sMAP-specific probe, MASP-2 H-chain , MASP-2 L chain or the neo gene. A specific parane range (2.2 kb) was observed in homozygous (- / -) mice. (B) Quantitative RT-PCR. MASP-2 H and L chains and desMAP cDNA fragments were amplified by real-time PCR in a LightCycler instrument (Roche Diagnostics). Synthesized cDNAs from wild type and homozygous (- / -) mouse (+ / +) mouse liver RNAs were used as standard. The data shown are the averages of two experiments.

FIGURE 22: MASP-2 deficiencies in homozygous (- / -) mouse serum. (A) Immunomancening of MASP-2 esMAP in mouse serum. Serum from wild type (+ / +) mice or homozygous (- / -) mice (2 μl) were immunoblotted and detected with anti-MASP-2 / sMAP antiserum. (B) Detection of esMAP MASPs in the MBL-MASP-sMAP complex. Mouse serum was incubated with agarose mannan-gel and sMAP, MASP-I and MASP-2 in the gel-bound MBL complex were detected as described in Materials and Methods.

FIGURE 23: Decreased cleavage of C4 and C3 in homozygous (- / -) mouse serum. (A) Deposition of C4 in mannan-coated reservoirs. Mouse serum was diluted 2-fold and incubated in mannan-coated reservoirs at room temperature for 30 min. After washing the reservoirs, human C4 was added to each reservoir and incubated on ice for 30 min. The amount of human C4 deposited in the reservoirs was measured using HRP-conjugated anti-C4 human polyclonal antibody. (B) Deposition of C3 in mannan-coated reservoirs. Diluted mouse serum was added to the mannan coated reservoirs and incubated at 37 ° C for 1 h. Endogenous C3 deposition in the reservoirs was detected with HRP-conjugated anti-human C3c polyclonal antibody.

FIGURE 24: Competitive binding of sMAP and MASP-2 aMBL. (A) Reconstitution of MBL-MASP-sMAP complex in homozygous mouse serum (- / -). MASP-2Í and / or rsMAP (4 μg) were incubated with homozygous (- / -) mouse serum (20 μl). The mixture was further incubated with agarose mannan-gel and rsMAP and MASP-2I in the gel-bound fraction were detected by immunoblotting. (B) Several quantities of MASP-2 (0 to 5 μg) and a constant amount of rsMAP (5 μg) were incubated with homozygous (- / -) mouse serum (20 μl) and further incubated with agarose mannan gel. (C) A constant amount of MASP-2 (0.5 μg) and various amounts of rsMAP (0 to 20 μg) were incubated with homozygous (- / -) mouse serum (20 μl). (D) Several quantities of rsMAP (0 to 20 μ§) were incubated with wild type (+ / +) mouse serum (20 μl).

FIGURE 25: Restoration of C4 deposition activity by the addition of rMASP-2. Various amounts of rsMAP (0 to 5 μg) (A) ourMASP-2 (0 to 1.5 μg) (B) were incubated with 0.5 μΐ homozygous (- / -) mouse serum in a total volume of 20 μΐ in TBS-Ca2 + buffer on ice overnight. Then the mixture was diluted with 80 μΐ of TBS-Ca2 + / BSA buffer and added to the mannan coated reservoirs and the amount of C4 deposited in the reservoirs was measured.

FIGURE 26: Reduction of C4 deposition activity by sMAP addition. (A) rMASP-2 (1 μg) and various amounts of rsMAP (0 to 0.5μg) were incubated with 0.5 μΐ of homozygous (- / -) mouse serum.The mixture was added to the mannan-coated reservoirs and The amount of C4 deposited in the reservoirs was measured. (B) rsMAP (0 to 0.7μg) was incubated with wild-type serum (0.5 μΐ) and the amount of C4 deposited in mannan-coated reservoirs was measured.

RESULTS:

SMAP and MASP-2 expression in homozygous mice (- / -)

To clarify the role of sMAP in vivo, we have established a mouse targeted gene that lacks sMAP. A bleach vector was constructed to replace the sMAP-specific exon (exon5) with a neomycin resistance gene cassette (Figure 20A). Positive ES clones were injected into C57BL / 6 blastocysts and the breeder's chimeras crossbreed with C57BL / 6J females. Southern blot DNA analysis of the tail of agouti puppies shows germline transmission of the targeted allele (Figure 20B). Heterozygous (+/-) mice were screened by Southern blot analysis of BamH-digested tail DNA using the probe indicated in FIGURE 20A. Southern blot analysis showed 6.5 kbp and 11 kbp bands in the DNA of heterozygous (+/-) mice (Figure 20B). Heterozygous (+/-) mice were backcrossed with C57BL / 6J mice. To obtain homozygous (- / -) mice, heterozygous (+/-) mice were intercrossed. Mouse mice (- / -) (C57BL / 6J fundamentals) were identified by PCR based on tail DNA genotyping, yielding a single range of 1.8 kbp (Figure 20C).

Homozygous (- / -) mice developed normally and showed no significant difference in the body weight of wild type (+ / +) mice. There was no morphological difference between each of them. In a Northern blot analysis, the sMAP-specific probe detected a single 0.9 kb band in wild type (+ / +) mice, whereas no specific band was detected in homozygous (- / -) mice (Figure 2IA). When the MASP-2 H or L chain specific probe was used, several specific strains were detected in previously reported wild type (+ / +) mice (Stover et al, 1999) and the H chain specific probe also detected the specific range. from sMAP. However, in mice (- / -) the corresponding ranges were very weak and several extra ranges were detected. We also performed a quantitative RT-PCR analysis to check sMAP eMASP-2 mRNA expression levels. In homozygous (- / -) mice, the desMAP mRNA expression was completely abolished and that of MASP-2 was also decreased sharply: it was quantified as about 2% of that wild type (+ / +) decamines in both the H chain and in L by real-time PCR (Figure 21B). In addition, we examined the expression of deMASP-2 at protein level. Both sMAP and MASP-2 were undetectable in the serum of homozygous (- / -) mice by immunoblotting (Figure 22A). Following incubation of the homozygous (- / -) mouse serum with agarose mannan gel, both sMAP and MASP-2 were not detectable in the gel-bound fraction, although MASP-I was detected in the complex (Figure 22B).

C4 and C3 cleavage activities through lectin pathway in homozygous mouse serum (- / -)

When homozygous (- / -) mouse serum was incubated in mannan-coated reservoirs, the amount of human C4 deposited in the reservoirs was about 20% of that in seronormal dilutions ranging from 1/400 to 1/50 (Figure 23A). We also examined the C3 deposition activity of the lectin pathway in homozygous (- / -) mouse serum. Mouse serum was added to the mannan-coated reservoirs and the amount of endogenous C3 deposited in the reservoirs was measured. The amount was decreased in serodeficient and was 21% of that in normal serum at a 1/10 dilution (Figure 23B).

Reconstitution of MBL-MASP-sMAP complex in mouse serum - (- / -)

When recombinant sMAP (rsMAP) or inactive mutant MASP-2 mouse (MASP-2I) mice were added to the homozygous (- / -) mice, both recombinants were able to bind to MBL (Figure 24A, lines 3 and 4). When rsMAP and MASP-2I were simultaneously incubated with serum (Figure 24A, line 5), both recombinants were detected in the MBL-MASP-sMAP complex.

However, the amount of sMAP bound to the complex was less than when only rsMAP was incubated with serum. We then further investigate the competitive binding of sMAP and MASP-2 to MBL. A constant amount of rsMAP and various amounts of MASP-23 were added to the deficient serum. The binding of rsMAP decreased in a data-dependent manner with increasing amounts of MASP-2 (Figure 24B). Conversely, the amount of MBL-bound MASP-21 decreased by the addition of rsMAP (Figure 24C). When rsMAP was added to wild-type serum, binding of both endogenous sMAP and MASP-2 to MBL decreased in a dose-dependent manner (Figure 24D).

Reconstitution of C4 deposition activity in mouse serum - (- / -)

We performed a C4 deposition reconstitution experiment in mannan-coated reservoirs using recombinant. When rsMAP was added to the deficient serum, the amount of deposited C4 actually decreased to basal levels in a dose dependent manner (Figure 25A). When rMASP-2 was added to the serum, the amount of C4 was restored to up to 46% of that of wild type serum in a dose dependent manner and reached a plateau (Figure 25B). Next, we investigate the effect of sMAP on C4 deposition. When a constant amount of rMASP-2 and various amounts of dersMAP were added to the deficient serum, the amount of deposited C4 decreased with the addition of rsMAP in a dose-dependent manner (Figure 26A) and the addition of rsMAP to wild-type serum also decreased. amount of C4 deposited (Figure 26B), suggesting that sMAP plays a regulatory role in activating the lectin pathway.

Debate

We generate sMAP - / - mice through targeted disruption of sMAP specific exon. The MASP-2 expression level was also greatly decreased at both mRNA and deprotein levels in these mice (Figures 21 and 22). A Northern blot analysis with a MASP-2 probe showed only extra bands in the poly (A) + RNA of sMAP - / - mice, suggesting that the normal junction of the MASP-2 gene was altered by sMAP targeting and therefore the level of expression of MASP-2. -2 has been markedly decreased. As a result, the cleavage of C4 by MBL-MASP complex in deficient serum was decreased by about 80% compared with that in normal serum (Figure 23A). In the reconstitution experiments, C4 cleavage activity was restored by the addition of derMASP-2 but not rsMAP (Figure 25). The reduction in C4 deposition observed in deficient serum should be caused by MASP-2 deficiencies in the MBL-MASP complex (Figure 22B). Therefore, it is clear that MASP-2 is essential for C4 activation by the MBL-MASP complex. However, the addition of rMASP-2 does not completely restore cleavage activity and C4 uptake has reached a plateau. As reported previously (Cseh et al, J Immunol 169: 5735-5743 (2002); Iwaki and Fujita, J Endotoxin Res 11: 47-50 (2005), most rMASP-2 was converted to active form by autoactivation during purification procedures and some have lost their protease activity.As active or inactive states of MASP-2 have no significant influence on their association with MBL (Zundel etal, J Immunol 172: 4342-4350 (2004), it is possible that rMASP-2 that lost its protease activity binds to MBL and competitively prevents active form association, thus resulting in an incomplete restoration of C4 deposition. C3 cleavage activity of the dalectin pathway was also attenuated in deficient serum (Figure 23Β) The decline in deposited C3 amount is probably due to the very low reactivity level of C3 convertase, which consists of C4b and C2a fragments generated by MASP-2.

MASP and sMAP each associated as ecomplex homodimers formed with MBL or L-phytoline through their N-terminal CUB and EGF equivalent domains (Chen and Wallis, J Biol Chem 276: 25894-25902 (2001); Cseh et al, J Immunol 169: 5735-5743 (2002); Thielenset al, J Immunol 166: 5068-5077 (2001); Zundel et al, J Immunol 172: 4342-4350 (2004)). The crystal structures of MASP-2 sMAP and CUBl-EGF-CUB2 segment reveal their homodimeric structure (Feinberg et al, EMBO J 22: 2348-2359 (2003); Gregory et al, J Biol Chem 278: 32157-32164 (2003)). The collagen equivalent domain of MBL is involved in association with MASPs (Wallis and Cheng, J Immunol 163: 4953-4959 (1999); Wallis and Drickamer, J Biol Chem 279: 14065-14073 (1999) and some mutations introduced in the domain have decreased. MBL binding to MASP-I and MASP-2 CUB1-EGF-CUB2 segments (Wallis and Dodd, J BiolChem 275: 30962-30969 (2000).) The binding sites for MASP-2 and paraMASP-1/3 se overlap but are not identical (Wallis et al, J Biol Chem279: 14065-13073 (2004).) Although the MBL sMAP binding site has not yet been identified, the binding sites for sMAP and MASP-2 are probably identical because CUBl-EGF region is the same in sMAP eMASP-2, so it is reasonable that sMAP and MASP-2 compete with each other to paralyze MBL in the MBL-MASP-sMAP complex reconstitution (Figure 24) .The sMAP affinity for MBL is lower than that of MASP-2 (Csehet al, J Immunol 169: 5735-5743 (2002); Thielens et al, J Immunol 166: 5068-5077 (2001)). sMAP concentration in mouse serum has not been determined. As shown in FIGURE 22A, however, the amount of sMAP in wild-type serum is much higher than that of MASP-2. Therefore sMAP is able to occupy the binding site of MASP-2 / sMAP and prevent MASP-2 from binding. MBL and therefore the C4-declining activity of the MBL-MASP complex is reduced. The regulatory mechanism of sMAP in the lectin pathway remains to be investigated. It is still unknown whether sMAP plays its regulatory role before or after complement activation. sMAP may prevent inadvertent activation of the MBL-MASP complex prior to microbial infection or suppress excessive activation of the lectin pathway once activated. There is another potential regulator in the lectin pathway. MASP-3 is also a MASP-2 competitor in binding to MBL and down-regulates MASP-2 C4 and C2 declining activity (Dahl et al, Immunity 15: 127-135 (2001)). Although the interaction between sMAP and MASP-3 has not been investigated, as they may be able to co-regulate dalectin pathway activation cooperatively.

In this report we demonstrate that sMAP and MASP-2 compete to bind MBL and sMAP has the ability to downregulate the lectin pathway, which is activated by the MBL-MASP complex. It is reasonable that sMAP also regulates another pathway of the activated lectin pathway with the phycoline-MASP complex. MASP-2 and sMAP are also competitors to paralyze mouse ficoline A and down-regulate the C4 cleavage activity of ficoline A-MASP complex (Y Endo et al, in preparation). A study of MBL null mice has recently been reported (Shi et al, JExp Med 199: 1379-1390 (2004). MBL null mice have no C4 cleavage activity in the MBL pathway of lectin and are susceptible to Staphylococcus aureus infections. In the present study, sMAP - / - mice, which are also MASP-2 deficient, showed reductions in C3 cleavage activity in addition to C4 cleavage activity in the lectin pathway, because of their impaired opsonization activity, sMAP deficient mice may be susceptible to bacterial infections Further investigation of sMAP deficient mice will clarify the role of the lectin pathway in protecting against infectious diseases.

Another important finding is that the addition of rsMAP to normal serum results in a reduction in C4 activation (Figure 26B). The lectin pathway has also been shown to regulate inflammation and tissue damage in various organs (de Vries et al, Am J Pathol 165: 1677-1688 (2004); Fiane et al, Circulation 108: 849-856 (2003); Jordan et al , Circulation 104: 1413-1418 (2001); Walsh et al, J Immunol 175: 541-546 (2005)). In MBL-deficient patients undergoing treatment for abdominal thoracic aortic aneurysm, complement was not activated and proinflammatory marker levels were reduced following surgery (Fiane etal, Circulation 108: 849-856 (2003)). Accumulated evidence has demonstrated the potential pathophysiological role of MBL during ischemia and reperfusion conditions in a variety of vascular beds. Therefore, MBL-specific obliteration or dalectin complement pathway inhibition may represent a therapeutically relevant strategy for the prevention of ischemia / perfusion associated injury. Thus, it is possible that MAP may be a candidate for such an inhibitor, since it acts as an attenuator of the lectin activation pathway.

EXAMPLE 31

This example demonstrates that MASP-2 is responsible for activating C4 offset C4.

Rationale / Rational analysis: More recently, it has been shown that inhibiting the alternative pathway protects the kidney from acute ischemic failure (Thurman et al., J. Immunol 170: 1517-1523 (2003)). The data described herein imply that the lectin pathway instructs activation of the alternative pathway, which in turn amplifies the activation of complementosergistically. We hypothesized that transient inhibition of lectin pathway may also affect alternate pathway activation and thus improves long-term organ transplant outcome by limiting lesion and complement-mediated graft inflammation and may moderate unwanted induction of a pathway. adaptive immune response against the graft and reduces the risk of secondary graft rejection through the adaptive immune system. This is supported by recent clinical data showing that a partially impaired lectin pathway resulting from inherited MBL deficiencies (present in about 30% of the human population) is associated with increased renal halograft survival in humans (Berger, Am J Transplant 5 : 1361-1366 (2005)).

Invasion of component C3 and C4 nalesion by ischemia-reperflection (I / R) has been well established in models of transient intestinal and muscular ischemia using gene-targeted mouse strains (Weiser et al., J Exp Med 183: 2342-2348 ( Williamset et al., Appl Appliol 86: 938-42 (1999)). It is well established that C3 has a prominent role in renal I / R injury and secondary graft rejection (Zhouet al., J Clin Invest 105: 1363-1371 (2000); Pratt et al., Nat Med 8: 582-587 (2002). ); Farrar, et al., Am J. Pathol 164: 133-141 (2004)). It was therefore surprising that a phenotype for C4 deficiencies was not observed in the published models of mouse renal allograft rejection (Lin, 2005 In press). Subsequent analysis of C4-deficient sera and plasma from non-C-deficient mice, however, indicated that these mice retain residual functional activity showing C3-dependent cleavage and complement downstream activation (see FIGURE 27C).

The existence of a functional C4 shift (and C2 shift) is a phenomenon previously described (but not fully characterized) by various investigators (Miller et al., Proc Natl Acad Sci 72: 418-22 (1975); Knutzen Steuer et al., J Immunol 143 (7): 2256-61 (1989); Wagner et al., J Immunol 163: 3549-3558 (1999) and relates to the alternative-independent pathway of C3 metabolization in C4 (and C2) deficient sera.

Methods: Effects of the lectin pathway and the classical pathway on C3 deposition. Mouse plasma (with EGTA / Mg as anticoagulant) was diluted and recalcified in 4.0 mM barbital, 145 mM NaCl, 2.0 mM CaCl 2, 1.0 mM MgCl 2, pH 7.4, then added. to mannan-coated microtiter plates (as shown in FIGURE 27A and 27C) or zymosan (as shown in FIGURE 27B) and incubated for 90 min at 37 ° C. The plates were washed 3 times with 10 mM Tris-Cl, 140 mM 5.0 mM NaCl5, 0.05% Tween 20, pH 7.4 then C3b uptake was measured using an anti-mouse C3c antibody.

Results: The results shown in FIGURE 27A-C are representative of 3 independent experiments. When using the same ones in reservoirs coated with immunoglobulin complexes instead of mannan or zymosan, C3b deposition and Factor B cleavage are observed in pooled WT (+ / +) sera and MASP-2 (- / -) sera. but not in depleted Clq sera (data not shown). This indicates that alternate path activation can be restored in serosMASP-2 - / - when the initial C3b is provided through the CP activity. FIGURE 27C represents the surprising discovery that C3 can be efficiently activated in a C4-deficient plasma lectin pathway-dependent manner (- / -). This "C4 shift" is abolished by inhibiting lectin pathway activation by preincubation of soluble mannan or mannose plasma.

It can be observed that C3b deposition in ezimosan manana is severely compromised in MASP-2 (- / -) deficient mice, even under experimental conditions which according to long-published alternative pathway activation must be permissive for all three pathways. . As shown in FIGURE 27A-C, MASP-2 (- / -) deficient mouse plasma do not activate C4 via the lectin pathway and do not cleave C3, either through the lectin pathway or the alternate pathway. We therefore hypothesize that MASP-2 is required in this C4 deviation. Further progress in identifying components likely to be involved in the lectin pathway-dependent C4 shift has been most recently reported by Prof. Teizo Fuj ita. Plasma from C4-deficient mice crossed with the Fujita MASP-1/3-deficient mouse strain loses the residual ability of C4-deficient plasma to cleave C3 via the lectin pathway. This was restored by the addition of recombinant MASP-I to the combined C4 and MASP-1/3 deficient plasmas (Takahashi, Mol Immunol 43: 153 (2006), suggesting that MASP-1 is involved in the formation of C3-cleaved lectin pathway complexes). in the absence of C4 (recombinant MASP-1 does not cleave C3, but cleaves C2; Rossi et al., J Biol Chem 276: 40880-7 (2001); Chen et al., J BiolChem 279: 26058-65 (2004). We observe that MASP-2 is required for this deviation to be formed.

Although more functional equative parameters and histology are required to consolidate this pilot study, its preliminary results provide strong support for the hypothesis that activation through the lectin pathway contributes significantly to the pathophysiology of renal I / R injury as seen in MASP-2- mice. / - show a much faster recovery from renal functions.

Although the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes may be made to this point without departing from the spirit and scope of the invention.

Embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: SEQUENCE LISTING

<110> Schwaeble, H.W.Stover, C.M.Tedford, C.E.Parent, J.B.Fujita, T.

<120> METHODS TO TREAT CONDITIONS ASSOCIATED WITH ACTIVATION OF

MASP-2 DEPENDENT COMPLEMENT <130> OMER-1-292OO <150> US 11 / 645,359 <151> 2006-12-22 <150> US 60 / 788,876 <151> 2006-04-03 <150> US 60 / 578,847 <151> 2004-06-10 <150> US 11 / 150,883 <151> 2005-06-09 <160> 65

<170> PatentIn version 3.2

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290 295 300

Ala Pro Pro Asn Gly His Val Pro Valn Gln Ala Lys Tyr Ile Leu305 310 315 320Lys Asp Ser Phe Ser Ile Phe Cys 325 Gly His Leu Pro Leu Lys Ser Phe 340 Ser Trp Asp Arg Pro Ala 355 360Pro Asp Asp Leu Pro Ser Gly Arg 370 375 Val Thr Thr Tyr Lys Wing Val Ile385 390 Tyr Thr Met Lys Val Asn Asp Gly 405 Phe Trp Being Ser Lys Gly Glu 420 Val Cys Gly Leu Ser Being Arg Thr 435 440Gln Lys Ala Lys Pro Gly Asp Phe 450 455 Gly Thr Thr Wing Gly Wing Leu465 470 Wing His Wing Val Tyr Glu Wing Gln 485 Ile Arg Met Gly Thr Leu Lys Arg 500 Trp Ser Glu Wing Val Phe Ile His 515 520Phe Asp Asn Asp Ile Wing Leu Ile 530 535 Asn Ser Asn Ile Thr Pro Ile Cys545 550 Phe Met Arg Thr Asp Ile Gly 565 Gln Arg Gly Phe Leu Wing Arg Asn 580 Val Asp His Gln Lys Cys Thr Wing 595 600Arg Gly Be Val Thr Wing Asn Met 610 615 Gly Lys Asp Be Cys Arg Gly Asp625 630 Asp Be Glu Thr Glu Arg Trp Phe 645 Be Met Asn Cys Gly Glu A la Gly 660 Ile Asn Tyr Ile Pro Trp Ile Glu 675 680 <210> 6 <211> 671 <212> PRT <213> Homo sapien <4 00> 6 Thr Pro Read Gly Pro Lys Trp Pro1 5 Ser Pro Gly Phe Pro Gly Glu Tyr 20 Thr Leu Thr Ala Pro Pro Gly Tyr 35 40Phe Asp Leu Glu Leu Be His Leu 50 55 Ser Ser Gly Alys Lys Val Leu Ala65 70 Asp Thr Glu Arg Ala Pro Gly Lys 85

Glu Thr Gly Tyr Glu Leu Leu Gln 330 335 Thr Wing Val Cys Gln Lys Asp Gly345 350 Cys Ser Ile Val Asp Cys Gly Pro 365 Val Glu Tyr Ile Thr Gly Pro Gly 380 Gln Tyr Ser Cys Glu Thr Phe 395 400Lys Tyr Val Cys Glu Wing Asp Gly 410 415 Lys Ser Leu Pro Val Cys Glu Pro425 430 Thr Gly Gly Arg Ile Tyr Gly 445 Pro Trp Gln Val Leu Ile Leu Gly 460 Leu Tyr Asp Asn Trp Val Leu Thr 475 480Lys His Asp Wing 490 495 Leu Be Pro His Tyr Thr Gln Ala505 510 Glu Gly Tyr Thr His Asp Ala Gly 525 Lys Leu Asn Asn Lys Val Val Ile 540 Leu Pro Arg Lys Glu Ala Glu Ser 555 560Thr Ala Ser Gly Trp Gly Leu Thr 570 575 Leu Met Tyr Val Asp Ile Pro Ile585 590 Wing Tyr Glu Lys Pro Tyr Pro 605 Leu Cys Wing Gly Leu Glu Ser Gly 620 Ser Gly Gly Wing Leu Val Phe Leu 635 640Val Gly Gly Ile Val Ser Trp Gly 650 655 Gln Tyr Gly Val Tyr Thr Lys Val665 670 Asn Ile Ile Ser Asp Phe 685 Glu Pro Val Phe Gly A rg Leu Wing 10 15 Wing Asn Asp Gln Glu Arg Arg Trp25 30 Arg Leu Arg Leu Tyr Δ S Phe Thr HisCys Glu Tyr Asp Phe Val Lys Leu 60 Thr Read Cys Gly Gln Glu Ser Thr 75 80Asp Thr Phe Tyr Ser Leu Gly Ser Qn QRSer Read Asp Ile Thr Phe Arg Be Asp Tyr Be Asn Glu Lys Pro Phe 100 105 110 Thr Gly Phe Glu Wing Phe Tyr Wing Glu Asp Ile Asp Glu Cys Gln 115 120 125 Val Wing Pro Gly Glu Ala Pro Thr Cys Asp His Cys His Asn His 130 135 140 Leu Gly Gly Phe Tyr Cys Ser Cys Arg Wing Gly Tyr Val Leu His Arg145 150 155 160Asn Lys Arg Thr Cys Ser Wing Leu Cys Ser Gly Gln Val Phe Thr Gln 165 170 175 Arg Ser Gly Glu Leu Ser Be Pro Glu Tyr Pro Arg Pro Tyr Pro Lys 180 185 190 Leu Be Ser Cys Thr Tyr Ser Ile Ser Be Glu Glu Gly Phe Ser Val 195 200 205 Ile Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Thr 210 215 220 Leu Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr Asp Arg Glu Glu 235 240Gly Pro Phe Cys Gly Lys Thr. Leu Pro His Arg Ile Glu Thr Lys Ser 245 250 255 Asn Thr Val Thr Ile Thr Phe Val Thr Asp Glu Ser Gly Asp His Thr 260 265 270 Gly Trp Lys Ile His Tyr Thr Wing Thr Cys Pro Tyr Pro 275 280 285 Met Pro Wing Pro Asn Gly His Val Pro Pro Val Gln Wing Lys Tyr Ile 290 295 300 Leu Lys Asp Ser Phe Ser Ile Phe Cys Glu Thr Gly Tyr Glu Leu Leu305 310 315 320Gln Gly His Leu Pro Read Lys Ser Phe Thr Ala Val Cys Gln Lys Asp 325 330 335 Gly Be Trp Asp Arg Pro Met Pro Wing Cys Be Ile Val Asp Cys Gly 340 345 350 Pro Pro Asp Asp Leu Pro Be Gly Arg Val Glu Tyr Ile Thr Gly Pro 355 360 365 Gly Val Thr Thr Tyr Lys Val Ile Wing Gln Tyr Be Cys Glu Glu Thr 370 375 380 Phe Tyr Thr Met Lys Val Asn Asp Gly Lys Tyr Val Cys Glu Wing Asp385 390 395 400Gly Phe Trp Thr Be Lys Gly Glu Lys Ser Leu Pro Val Cys Glu 405 410 415 Pro Val Cys Gly Read Be Wing Arg Thr Thr Gly Gly Arg Ile Tyr Gly 420 425 430 Gly Gln Lys Lys Pro Wing Gly Asp Phe Pro Trp Gln Val Leu Ile Leu 435 440 445 Gly Gly Thr Thr Wing Ala Gly Wing Leu Tyr Asp Trp Val Leu 450 455 460 Thr Wing His His Val Tyr Glu Gln Lys His Asp Wing To Be Wing Leu465 470 475 480Asp Ile Arg To Met Gly Thr Leu Lys Arg To Read Pro To His Tyr Thr Gln 485 490 495 Wing To Trp To Be Glu Wing Val Phe Ile To His Glu Gly Tyr Thr To His Wing 500 505 510 Gly Phe Asp Asn Asp Ile Wing Leu Ile Lys Leu Asn Asn Lys Val Val 515 520 525 Ile Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys Glu Wing Glu 530 535 540 Ser Phe Met Arg Thr Asp Ile Gly Thr Wing To Be Gly Trp Gly Leu545 550 555 560Thr Gln Arg Gly Phe Leu Arg Wing Asn Leu Met Tyr Val Asp Ile Pro 565 570 575 Ile Val Asp His Gln Lys Cys Thr Wing Tyr Glu Lys Pro Tyr 580 585 590 Pro Arg Gly Be Val Thr Ala Asn Met Leu Cys Wing Gly Leu Glu Ser595 600

Gly Gly Lys Asp Being Cys Arg Gly

610 615

Read Asp Be Glu Thr Glu Arg Trp625 630

Gly Ser Met Asn Cys Gly Glu Ala645

Val Ile Asn Tyr Ile Pro Trp Ile660

<210> 7 <211> 4900 <212> DNA <213> Homo sapien <400> 7

cctgtcctgc ctgcctggaa ctctgagcag gctggagtca tggagtcgat tcccagaatc 60

ccagagtcag ggaggctggg ggcaggggca ggtcactgga caaacagatc aaaggtgaga 120

ccagcgtagg actgcagacc aggccaggcc agctggacgg gcacaccatg aggtaggtgg 180

gcgccacagc ctccctgcag ggtgtggggt gggagcacag gcctgggcct caccgcccct 240

gccctgccca taggctgctg accctcctgg gccttctgtg tggctcggtg gccaccccct 300

taggcccgaa gtggcctgaa cctgtgttcg ggcgcctggc atcccccggc tttccagggg 360

agtatgccaa tgaccaggag cggcgctgga ccctgactgc accccccggc taccgcctgc 420

gcctctactt cacccacttc gacctggagc tctcccacct ctgcgagtac gacttcgtca 480

aggtgccgtc agacgggagg gctggggttt ctcagggtcg gggggtcccc aaggagtagc 540

cagggttcag ggacacctgg gagcaggggc caggcttggc caggagggag atcaggcctg 600

ggtcttgcct tcactccctg tgacacctga ccccacagct gagctcgggg gccaaggtgc 660

tggccacgct gtgcgggcag gagagcacag acacggagcg ggcccctggc aaggacactt 720

tctactcgct gggctccagc ctggacatta ccttccgctc cgactactcc aacgagaagc 780

cgttcacggg gttcgaggcc ttctatgcag ccgagggtga gccaagaggg gtcctgcaac 840

atctcagtct gcgcagctgg ctgtgggggt aactctgtct taggccaggc agccctgcct 900

tcagtttccc cacctttccc agggcagggg agaggcctct ggcctgacat catccacaat 960

gcaaagacca aaacagccgt gacctccatt cacatgggct gagtgccaac tctgagccag 1020

ggatctgagg acagcatcgc ctcaagtgac gcagggactg gccgggcgcg gcagctcacg 1080

cctgtaattc cagcactttg ggaggccgag gctggcttga taatttgagg gtcaggagtt 1140

caaggccagc cagggcaaca cggtgaaact ctatctccac taaaactaca aaaattagct 1200

gggcgtggtg gtgcgcacct ggaatcccag ctactaggga ggctgaggca ggagaattgc 1260

ttgaacctgc gaggtggagg ctgcagtgaa cagagattgc accactacac tccacctggg 1320

cgacagacta gactccgtct caaaaaacaa aaaacaaaaa ccacgcaggg ccgagggccc 1380

atttacaagc tgacaaagtg ggccctgcca gcgggagcgc tgcaggatgt ttgattttca 1440

gatcccagtc cctgcagaga ccaactgtgt gacctctggc aagtggctca atttctctgc 1500

tccttagaag ctgctgcaag ggttcagcgc tgtagccccg ccccctgggt ttgattgact 1560

cccctcatta gctgggtgac ctcggccgga cactgaaact cccactggtt taacagaggt 1620

gatgtttgca tctttctccc agcgctgctg ggagcttgca gcgaccctag gcctgtaagg 1680

tgattggccc ggcaccagtc ccgcacccta gacaggacct aggcctcctc tgaggtccac 1740

tctgaggtca tggatctcct gggaggagtc caggctggat cccgcctctt tccctcctga 1800

cggcctgcct ggccctgcct ctcccccaga cattgacgag tgccaggtgg ccccgggaga 1860

ggcgcccacc tgcgaccacc actgccacaa ccacctgggc ggtttctact gctcctgccg 1920

cgcaggctac gtcctgcacc gtaacaagcg cacctgctca ggtgagggag gctgcctggg 1980

ccccaacgca ccctctcctg ggatacccgg ggctcctcag ggccattgct gctctgccca 2040

ggggtgcgga gggcctgggc ctggacactg ggtgcttcta ggccctgctg cctccagctc 2100

cccttctcag ccctgcttcc cctctcagca gccaggctca tcagtgccac cctgccctag 2160

cactgagact aattctaaca tcccactgtg tacctggttc cacctgggct ctgggaaccc 2220

ctcatgtagc cacgggagag tcggggtatc taccctcgtt ccttggactg ggttcctgtt 2280

ccctgcactg ggggacgggc cagtgctctg gggcgtgggc agccccaccc tgtggcgctg 2340

accctgctcc cccgactcgg tttctcctct cggggtctct ccttgcctct ctgatctctc 2400

ttccagagca gagcctctag cctcccctgg agctccggct gcccagcagg tcagaagcca 24 60

gagccaggct gctggcctca gctccgggtt gggctgagat gctgtgcccc aactcccatt 2520

cacccaccat ggacccaata ataaacctgg ccccacccca cctgctgccg cgtgtctctg 2580

gggtgggagg gtcgggaggc ggtggggcgc gctcctctct gcctaccctc ctcacagcct 2640

catgaacccc aggtctgtgg gagcctcctc catggggcca cacggtcctt ggcctcaccc 2700

cctgttttga agatggggca ctgaggccgg agaggggtaa ggcctcgctc gagtccaggt 2760

ccccagaggc tgagcccaga gtaatcttga accaccccca ttcagggtct ggcctggagg 2820

agcctgaccc acagaggaga caccctggga gatattcatt gaggggtaat ctggtccccc 2880

gcaaatccag gggtgattcc cactgcccca taggcacagc cacgtggaag aaggcaggca 2940

605

Asp Ser Gly Gly 620 Ala Leu Val PhePhe Val Gly 635 Gly Ile Val Ser Trp 640Gly Gln 650 Tyr Gly Val Tyr Thr 655 LysGlu Asn Ile Ile Ser Asp Phe 665 670atgttggggc tcctcacttc ctagaggcct cacaactcaagggtggggtg gtggtatggg atggggacca agccttccttacaccccgcc ccgtggcagc agcatcacgt gttccagcgaagtcatgatc actgttgcct tgaacttcca agaacagcccgggaaagggg gtgatgagag atccttcttc cggatgttccctggtcttgg ctgggttcgg gtaggagacc catgatgaattggctgtaag ggaatttagg ggagctccga aggggcccttctcttttccc gaattcccag ggacccagga gagtgtccctcatccacccc cgccccccgc cctggcagag ctggtggaacctggggttgc gactctggct caggacacca ccacgctccctgtgcgctcc atcccgagtg ctgcctgttt cagctaaagcctctctaagc ggcccctcag ccatcgggtg ggtcgtttgggctggccacc tgcagggccc agcccaaccc agggatgcagtcccagtttc ctgctcccca aggcatccac cctgctgttgcacgccaagt cactcccctg cccttccctc cttccagcccacccagaggt ctggggagct cagcagccct gaatacccacagttgcactt acagcatcag cctggaggag gggttcagtgtccttcgatg tggagacaca ccctgaaacc ctgtgtccctctcctgggcc cctcatcttg tcccagatcc tcccccttcatcctgcagca tggcccccac cacgttccc g tcaccctcggctctatggag gtcaaggctg gggcttcgag tacaagtgtgcaccccaatc catggcctgg gttggcctca ttggctgtccgttacttccc tccgcccagg ccagacccag gcagctgctcttctcagatt caaacagaca gagaagaaca tggcccattccaggattgaa acaaaaagca acacggtgac catcacctttccacacaggc tggaagatcc actacacgag cacagtgagctggaagcgca gagctgcctc tctctggagt gcaaggagctgggcaggact aggaagggac accaggttta gtggtgctgaaaggggaagc acccgtgccc tcctcagcag cacccagcatccacccattc acccatcact catcttttac ccacccacccccctcatcct tccaaccatt catcaatcac ccacccatccccacccattc ttctacctac ccatcctatc catccatcctacccatcctt cgttcggtca tccatcatca tccatccatc <210> 8 <211> <212 > <213> <4 00> 8

Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys15 10

Pro Read Gly Pro Lys Trp Pro Glu Pro Val Phe

20 25

Pro Gly Phe Pro Gly Glu Tyr Wing Asn Asp Gln

35 40

Leu Thr Pro Wing Gly Tyr Arg Leu Arg Leu

50 55

Asp Leu Glu Leu Being His Leu Cys Glu Tyr Asp65 70 75

Ser Gly Alys Lys Val Leu Ala Thr Leu Cys Gly

85 90

Thr Glu Arg Wing Pro Gly Lys Asp Thr Phe Tyr

100 105

Read Asp Ile Thr Phe Arg Be Asp Tyr Be Asn

115 120

Gly Phe Glu Wing Phe Tyr Wing Wing

130 135

<210> 9 <211> 181 <212> PRT <213> Homo sapien <4 00> 9

Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys15 10

atgccccccagaaggatagaggaaggagagcagggcaaggtccaggaaccaaacttgggaaggctcgaggtcttcctctttcagtgctcttgggggtgtgctcaaagcaatttctgggtaatgtcccagcgtgcgagggctgtgctccggggccgtatcctcattctggaacgactttctgcccagctgctgaccccaccggaggcagagctgaaatgctcccagctttctgtgggaagagtcacagatgaagtgggctcgtagagtgtaggtctgaggccttcaccacttttgccactcatcctttgcctctatcagca

ctgcagctgggcccagcccacaccagactcgtcaaaacagagggggctggatcactggggagatgctcctcctgtgtgtcagcccctaccagtgagggccgagaaaccccggcctcagggcacatccctgtgatagagggccaggtcttccaaactctccctttgtggagcaaggtctggaccccctacttcttcaggtgtggggagggggaggaggtggatgagcttctcattgccccaaatcaggagaagatccttgggggctcttctagcagcttctcattcttcaaatccttctgtacacaaccattccttctacc

136PRT

Homo sapien

Gly ser

Gly arg

Glu Arg45

Tyr Phe60

Phe val

Gln Glu

To be read

Glu Lys125

Val Wing

15Leu Ala30

Arg Trp

Thr his

Lys Leu

Be thr95

Gly ser110

Pro phe

Thr

To be

Thr

Phe

Ser80Asp

To be

Thr

300030603120318032403300336034203480354036003660372037803840390039604020408041404200426043204380444045004560462046804740480048604900

Gly Ser Val Wing Thr15Pro Read Gly Pro Lys Trp Pro Glu Val Val Phe Gly Arg Leu Wing Ser 20 20 30 30 Pro Gly Phe Pro Gly Glu Val Wing Asn Asp Gln Glu Arg Arg Trp Thr 35 40 45 Leu Thr Wing Val Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe 50 55 60 Asp Leu Glu Leu Be His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser65 70 75 80Ser Gly Wing Lys Val Leu Wing Thr Leu Cys Gly Gln Glu Ser Thr Asp 85 90 95 Thr Glu Arg Wing Pro Gly Lys Asp Thr Phe Tyr Be Read Gly Ser Be 100 105 110 Leu Asp Ile Thr Phe Arg Be Asp Tyr Be Asn Glu Lys Pro Phe Thr 115 120 125 Gly Phe Glu Wing Phe Tyr Ala Glu Asp Ile Asp Glu Cys Gln Val 130 135 140 Gly Pro Wing Gly Glu Pro Wing Cys Asp His His Cys His Asn His Leu145 150 155 160Gly Gly Phe Tyr Cys Be Cys Arg Wing Gly Tyr Val Leu His Arg Asn 165 170 175 Lys Arg Thr Cys Ser 180 <210 > 10 <211> 293 <212> PRT <21 3> Homo sapien <400> 10 Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Wing Thr1 5 10 15 Pro Leu Gly Pro Lys Trp Pro Val Phe Gly Leu Wing Ala Ser 20 25 30 Pro Gly Phe Pro Gly Glu Tyr Wing Asn Asp Gln Glu Arg Arg Trp Thr 35 40 45 Leu Thr Pro Wing Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe 50 55 60 Asp Leu Glu Leu Being His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser65 70 75 80Ser Gly Lys Wing Val Leu Thr Wing Read Cys Gly Gln Glu Ser As Thr 85 90 95 Glu Arg Wing Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser 100 105 110 Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr 115 120 125 Gly Phe Glu Wing Phe Tyr Wing Glu Asp Ile Asp Glu Cys Gln Val 130 135 140 Wing Pro Gly Glu Pro Thr Cys Asp His His Cys Asn His Leu145 150 155 160Gly Gly Phe Tyr Cys Be Cys Arg Wing Gly Tyr Val Read His Arg Asn Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gln Arg 180 185 190 Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro Tyr Pro Lys Leu 195 200 205 Ser Ser Cys Thr Tyr Ser Ile Ser Leu Glu Gly Phe Ser Val Ile 210 215 220 Leu Asp Phe Val Glu Be Phe Asp Val Glu Thr His Pro Glu Thr Leu225 230 235 240Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr Asp Arg Glu Glu His Gly 245 250 255 Pro His Arg Ile Glu Thr Lys Ser Asn 260 265 270 Thr Val Thr Ile Thr Phe Val Thr Asp

275 280 285Trp Lys Ile His Tyr 290 <210> 11 <211> 41 <212> PRT <213> Homo sapien <4 00> 11 Glu Asp Ile Asp Glu Cys Gln Val Wing Pro Gly Glu Wing Pro Thr Cys1 5 10 15 Asp His His Cys His Asn His Leu Gly Gly Phe Tyr Cys Ser Cys Arg 20 25 30 Wing Gly Tyr Val Leu His Arg Asn Lys 35 40 <210> 12 <211> 242 <212> PRT <213> Homo sapien <4 00> 12 Ile Tyr Gly Gly Gly Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp Gln Val1 5 10 15 Leu Ile Leu Gly Gly Thr Thr Wing Gly Wing Leu Leu Tyr Asp Asn 20 25 30 Trp Val Leu Thr Wing His His Val Tyr Glu Wing Gln Lys His Asp Ala 35 40 45 Ser Ala Read Asp Ile Arg Met Gly Thr Leu Lys Arg Leu Ser Pro His 50 55 60 Tyr Thr Gln Ala Trp Be Glu Val Val Phe Ile His Glu Gly Tyr Thr65 70 75 80His Asp Ala Gly Phe Asp Asn Asp Ile Wing Leu Ile Lys Leu Asn Asn 85 90 95 Lys Val Val Ile Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys 100 105 110 Glu Wing Glu Be Phe Met Arg Thr Asp Ile Gly Thr Wing Gly 115 120 125 Trp Gly Leu Thr Gln Arg Gly Phe Leu Wing Arg Asn Leu Met Tyr Val 130 135 140 Asp Ile Pro Ile Val Asp His Gln Lys Cys Thr Wing Ala Tyr Glu Lys145 150 155 160Pro Pro Tyr Pro Arg Gly Be Val Thr Wing Asn Met Leu Cys Ala Gly 165 170 175 Leu Glu Be Gly Gly Lys Asp Be Cys Arg Gly Asp Be Gly Gly Wing 180 185 190 Leu Val Phe Read Asp Be Glu Thr Glu Arg Trp Phe Val Gly Gly Ile 195 200 205 Val Ser Trp Gly Be Met Asn Cys Gly Glu Wing Gly Gln Tyr Gly Val 210 215 220 Tyr Thr Lys Val Ile Asn

Asp Phe

<210> 13

<211> 16

<212> PRT

<213> Artificial sequence <220>

<223> Synthetic peptide

<4 00> 13

Gly Lys Asp Being Cys Arg Gly Asp Wing Gly Gly Wing Leu Val Phe Leu15 10 15

<210> 14

<211> 15

<212> PRT

<213> Artificial sequence <220>

<223> Synthetic peptide <4 00> 14

Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu15 10 15

<210> 15 <211> 43 <212> PRT

<213> Artificial sequence <220>

<223> Synthetic peptide <4 00> 15

Pro Wing Thr Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe Asp15 10 15

Leu Glu Leu Being His Leu Cys Glu Tyr Asp Phe Val Lys Leu Being Ser

20 25 30

Gly Wing Lys Val Leu Wing Thr Thru Cys Gly Gln

35 40

<210> 16 <211> 8 <212> PRT <213> Artificial <220>

<223> Synthetic peptide <4 00> 16

Thr Phe Arg Be Asp

1 5

<210> 17

<211> 25

<212> PRT

<213> Artificial sequence <220>

<223> Synthetic peptide <4 00> 17

Phe Tyr Being Read Gly Being Read Asp Ile Thr Phe Tyr Being Asp Tyr15 10 15

Being Asn Glu Lys Pro Phe Thr Gly Phe20 25

<210> 18

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Synthetic peptide <4 00> 18

Ile Asp Glu Cys Gln Val Wing Pro Gly

1 5

<210> 19

<211> 25

<212> PRT

<213> Artificial sequence <220>

<223> Synthetic peptide <4 00> 19

Wing Asn Met Leu Cys Wing Gly Leu Glu Be Gly Gly Lys Asp Ser Cys15 10 15

Arg Gly Asp Ser Gly Gly Wing Leu Val20 25

<210> 20

<211> 960

<212> DNA

<213> Homo sapien

<220> <221> CDS <222> (51) .. (797) <400> 20attaactgag attaaccttc cctgagtttt ctcacaccaa ggtgaggacc atg tcc 56

Met ser1

ctg ttt cca tca ctc cct ctc ctc ctc agt atg gtg gca gcg tct 104Leu Phe Pro Ser Leu Leu Leu Leu Leu

5 10 15

tac tca gaa act gtg acc tgt gag gat gcc caa aag acc tgc cct gca 152Tyr Ser Glu Thr Val Cys Glu Asp Ala Gln Lys Thr Cys Pro Ala

20 25 30

gtg att gcc tgt age tct cca ggc till aac ggc ttc cca ggc aaa gat 200Val Ile Ala Cys Ser Ser Pro Gly Ile Asn Gly Phe Pro Gly Lys Asp

35 40 45 50

ggg cgt gat ggc acc aag gga gaa aag ggg gaa cca ggc caa ggg ctc 248Gly Arg Asp Gly Thr Lys Gly Glu Lys Gly Glu Pro Gly Gln Gly Leu

55 60 65

aga ggc tta cag ggc ccc cct gga aag ttg ggg cct cca gga aat cca 296Arg Gly Leu Gln Gly Pro Pro Gly Lys Leu Gly Pro Pro Gly Asn Pro

70 75 80

ggg cct tct ggg tca cca gga cca aag ggc caa aaa gga gac cct gga 344

Gly Pro Gly Gly Pro Gly Gly Pro Lys Gly Gly Lys Gly Asp Gly Pro

85 90 95

aaa agt ccg gat ggt gat agt age ctg gct gcc tca gaa aga aaa gct 392Lys Ser Pro Asp Gly Asp Ser Ser Leu Ala Ser Glu Arg Lys Ala

100 105 110

ctg caa aca gaa atg gca cgt up to aaa aag tgg ctc acc ttc tct ctg 440Leu Gln Thr Glu Met Ala Arg Ile Lys Lys Trp Leu Thr Phe Ser Leu

115 120 125 130

ggc aaa caa gtt ggg aac aag ttc ttc ctg acc aat ggt gaa ata atg 488Gly Lys Gln Val Gly Asn Lys Phe Phe Leu Thr Asn Gly Glu Ile Met

135 140 145

acc ttt gaa aaa gtg aag gcc ttg tgt gtc aag ttc cag gcc tct gtg 536Thr Phe Glu Lys Val Lys Alu Leu Cys Val Lys Phe Gln Ala Ser Val

150 155 160

gcc acc ccc agg aat gct gca gag aat gga gcc att cag aat ctc until 584Ala Thr Pro Arg Asn Wing Glu Wing Asn Gly Wing Ile Gln Asn Leu Ile

165 170 175

aag gag gaa gcc ttc ctg ggc till act gat gag aag aca gaa ggg cag 632Lys Glu Glu Ala Phe Leu Gly Ile Thr Asp Glu Lys Thr Glu Gly Gln

180 185 190

ttt gtg gat ctg aca gga aat aga ctg acc tac aca aac tgg aac gag 680Phe Val Asp Leu Thr Gly Asn Arg Leu Thr Tyr Thr Asn Trp Asn Glu

195 200 205 210

ggt gaa ccc aac aat gct ggt tct gat gaa gat tgt gta ttg cta ctg 728Gly Glu Pro Asn Asn Ala Gly Ser Asp Glu Asp Cys Val Leu Leu Leu

215 220 225

aaa aat ggc cag tgg aat gac gtc ccc tgc tcc acc tcc cat ctg gcc 776Lys Asn Gly Gln Trp Asn Val Asp Val Pro Cys Ser Thr Be His Leu Ala

230 235 240

gtc tgt gag ttc cct to tga agggtcatat cactcaggcc ctccttgtct 827Val Cys Glu Phe Pro Ile245

ttttactgca acccacaggc ccacagtatg cttgaaaaga taaattatat caatttcctc 887atatccagta ttgttccttt tgtgggcaat cactaaaaat gatcactaac agcaccaaca 947aagcaataat agt 960

<210> 21 <211> 248 <212> PRT <213> Homo sapien <400> 21

Met Ser Leu Phe Pro Ser Leu Pro Leu Leu Leu Read Met Val Val Wing 1 5 10 15 Wing Ser Tyr Ser 20 Glu Thr Val Cys 25 Glu Asp Wing Gln Lys 30 Thr CysPro Wing Val 35 Ile Cys Wing Ser Ser 40 Pro Gly Ile Asn Gly 45 Phe Pro GlyLys Asp 50 Gly Arg Asp Gly Thr 55 Lys Gly Glu Lys Gly 60 Glu Pro Gly GlnGly Read Arg Gly Leu Gln Gly Pro Pro Gly Lys Read Gly Pro Pro Gly65 70 75 80Asn Pro Gly Pro Pro Lys Gly Gln Lys Gly Asp 85 90 95 Pro Gly Lys Ser 100 Pro Asp Gly Asp Ser 105 Ser Leu Wing Wing Ser 110 Glu ArgLys Wing Leu 115 Gln Thr Glu Met Wing 120 Arg Ile Lys Trys 125 Leu Thr PheSer Leu 130 Gly Lys Gln Val Gly 135 Asn Lys Phe Phe Leu 140 Thr Asn Gly GluIle Met Thr Phe Glu Lys Val Lys Wing Leu Cys Val Lys Phe Gln Ala145 150 155 160Ser Val Ala Thr Pro 165 Arg Asn Wing Glu 170 Asn Gly Wing Ile Gln 175 AsnLeu Ile Lys Glu 180 Glu Phe Ward Leu Gly 185 Ile Thr Asp Glu Lys 190 Thr GluGly Gln Phe 195 Val Asp Leu Thr Gly 200 Asn Arg Leu Thr Tyr 205 Thr Asn TrpAsn Glu 210 Gly Glu Pro Asn Asn 215 Wing Gly Ser Asp Glu 220 Asp Cys Val LeuLeu Leu Lys Asn Gly Gln Trp Asn Asp Val Pro Cys Be Thr His225 230 235 240Leu Wing Val Cys Glu 245 Phe Pro Ile

<210> 22

<211> 6

<212> PRT

<213> Artificial sequence <220>

<223> Synthetic peptide consensus sequence <220>

<221> MISC_FEATURE

<222> (1). . (1)

<223> Where X at position 1 represents hydroxyproline <220>

<221> MISC_FEATURE

<222> (4) .. (4)

<223> Where X at position 4 represents hydrophobic residue

<4 00> 22

Xaa Gly Lys Xaa Gly Pro 5

<210> 23

<211> 5

<212> PRT

<213> Artificial sequence <220>

<223> Synthetic peptide <220>

<221> MISC_FEATURE

<222> (1). . (1)

<223> Where X represents hydroxyproline

<4 00> 23

Xaa Gly Lys Leu Gly1 5

<210> 24

<211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC FEATURE <222> (9) .. (15) <223> Where X in positions 9 and 15 represents hydroxyproline <400> 24 Gly Leu Arg Gly Leu Gln Gly Pro Xaa Gly Lys Leu Gly Pro Xaa Gly1 5 10 15 <210> 25 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Peptide synthetic <220> <221> MISC FEATURE <222> (3). (21) <223> Where X at positions 3, 6, 15, 21, 24, 27 represents hydroxyproline <400> 25 Gly Pro Xaa Gly Pro Xaa Gly Leu Arg Gly Leu Gln Gly Pro Xaa Gly1 5 10 15Lys Leu Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa 20 25 <210> 26 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> misc feature <222> (26). . (26) <223> Xaa can be any naturally occurring amino acid <220> <221> misc feature <222> (32). . (32) <223> Xaa can be any naturally occurring amino acid <220> <221> misc feature <222> (35) .. (35) <223> Xaa can be any naturally occurring amino acid <220> <221> misc feature <222> (41) .. (41) <223> Xaa can be any naturally occurring amino acid <220> <221> misc feature <222> (50) .. (50) <223> Xaa can be any naturally occurring amino acid <4 00> 26 Gly Lys Asp Gly Arg Asp Gly Thr Lys Gly Glu Lys Gly Glu Pro Gly1 5 10 15Gln Gly Leu Arg Gly Leu Gln Gly Pro Xaa Gly Lys Leu Gly Pro Xaa 20 25 30Gly Asn Xaa Gly Pro Be Gly Ser Xaa Gly Pro Lys Gly Gln Lys Gly 35 40 45Asp Xaa Gly Lys Ser 50 <210> 27 <211> 33 <212> PRT

<213> Artificial sequence <220>

<223> Synthetic peptide <220>

<221> MISC_FEATURE <222> (3) .. (33)

<223> Where X at positions 3, 6, 12, 18, 21, 30, 33 represents

hydroxyproline <400> 27

Gly Wing Xaa Gly Ser Xaa Gly Glu Lys Gly Wing Xaa Gly Pro Gln Gly1 5 10 15

Pro Xaa Gly Pro Xaa Gly Lys Met Gly Pro Lys Gly Glu Xaa Gly Asp20 25 30

Xaa

<210><211><212><213><220><223><220><221><222> <223>

2845PRT

Artificial sequenceSynthetic peptide

MISC_FEATURE (3) .. (45)

Where X at positions 3 hydroxyproline <400> 28

Gly Cys Xaa Gly Leu Xaa Gly Ala1 5

Thr Asn Gly Lys Arg Gly Glu Arg20

Gly Pro Wing Xaa Gly Pro Asn Gly35 40

2924PRT

Artificial sequence

6, 9, 27, 30, 36, 42, 45 represents

Xaa Gly Asp Lys Gly Glu Wing Gly

10 15

Gly Pro Xaa Gly Pro Xaa Gly Lys25 30

Wing Xaa Gly Glu Xaa45

<210><211><212><213><220><223> <400>

Synthetic peptide29

Leu Gln Arg Wing Leu Glu Ile Leu1 5

Asn Arg Pro Phe Leu Val Phe Ile20

<210> 30

<211> 559

<212> DNA

<213> Homo sapien

<400> 30

atgaggctgc tgaccctcct gggccttctgaagtggcctg aacctgtgtt cgggcgcctgaatgaccagg agcggcgctg gaccctgactttcacccact tcgacctgga gctctcccactcgggggcca aggtgctggc cacgctgtgccctggcaagg acactttcta ctcgctgggctactccaacg agaagccgtt cacggggttcgagtgccagg tggccccggg agaggcgcccggcggtttct actgctcctg ccgcgcaggctcagccctgt gctccggcc <210> 31 <211> 34

Pro Asn Arg Val Thr Ile Lys Ala10 15

tgtggctcgggcatcccccggcaccccccgctctgcgagtgggcaggagatccagcctgggaggccttctacctgcgacctacgtcctgc

tggccaccccgctttccagggctaccgcctacgacttcgtgcacagacacacattaccttatgcagccgaaccactgccaaccgtaacaa

cttgggcccgggagtatgccgcgcctctaccaagctgagcggagcgggccccgctccgacggacattgaccaaccacctggcgcacctgc

60120180240300360420480540559 <212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<400> 31

cgggcacacc atgaggctgc tgaccctcct gggc

<210> 32

<211> 33

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<400> 32

gacattacct tccgctccga ctccaacgag aag

<210> 33

<211> 33

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<400> 33

agcagccctg aatacccacg gccgtatccc aaa

<210> 34

<211> 26

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<400> 34

cgggatccat gaggctgctg accctc

<210> 35

<211> 19

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<400> 35

ggaattccta ggctgcata

<210> 36

<211> 19

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<400> 36

ggaattccta cagggcgct

<210> 37

<211> 19

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<400> 37

ggaattccta gtagtggat

<210> 38

<211> 25

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<400> 38

tgcggccgct gtaggtgctg tcttt

<210> 39 <211> 23

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<4 00> 39

ggaattcact cgttattctc gga

<210> 40

<211> 17

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<4 00> 40tccgagaata acgagtg

<210> 41

<211> 29

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<4 00> 41

cattgaaagc tttggggtag aagttgttc

<210> 42

<211> 27

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<4 00> 42

cgcggccgca gctgctcaga gtgtaga

<210> 43

<211> 28

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<4 00> 43

cggtaagctt cactggctca gggaaata

<210> 44

<211> 37

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<4 00> 44

aagaagcttg ccgccaccat ggattggctg tggaact

<210> 45

<211> 31

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<4 00> 45

cgggatcctc aaactttctt gtccaccttg g

<210> 46

<211> 36

<212> DNA

<213> Artificial sequence <220>

<223> Synthetic Oligo

<4 00> 46

aagaaagctt gccgccacca tgttctcact agctct <210> 47 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligo <400> 47cgggatcctt ctccctctaa cactct <210> 48 <211> 9 <213> PR <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 47cggatcctt ctccctctaa cactct <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 48Glu Pro Lys Ser Cys Asp Lys Thr His1 5 <210> 49 <211> 4960 <212> DNA <213> Homo Sapien <400> 49

ccggacgtgg tggcgcatgc ctgtaatccc agctactcgg gaggctgagg caggagaatt 60gctcgaaccc cggaggcaga ggtttggtgg ctcacacctg taatcccagc actttgcgag 120gctgaggcag gtgcatcgct ttggctcagg agttcaagac cagcctgggc aacacaggga 180gacccccatc tctacaaaaa acaaaaacaa atataaaggg gataaaaaaa aaaaaaagac 240aagacatgaa tccatgagga cagagtgtgg aagaggaagc agcagcctca aagttctgga 300agctggaaga acagataaac aggtgtgaaa taactgcctg gaaagcaact tctttttttt 360tttttttttt tttgaggtgg agtctcactc tgtcgtccag gctggagtgc agtggtgcga 420tctcggatca ctgcaacctc cgcctcccag gctcaagcaa ttctcctgcc tcagcctccc 480gagtagctgg gattataagt gcgcgctgcc acacctggat gatttttgta tttttagtag 540agatgggatt tcaccatgtt ggtcaggctg gtctcaaact cccaacctcg tgatccaccc 600accttggcct cccaaagtgc tgggattaca ggtataagcc accgagccca gccaaaagcg 660acttctaagc ctgcaaggga atcgggaatt ggtggcacca ggtccttctg acagggttta 720agaaattagc cagcctgagg ctgggcacgg tggctcacac ctgtaatccc agcactttgg 780gaggctaagg caggtggatc acctgagggc aggagttcaa gaccagcctg accaacatgg 840agaaacccca tccctaccaa tt aaataaaaaa agccaggt gtggtggtgc tcgcctgtaa 900tcccagctac ttgggaggct gaggtgggag gattgcttga acacaggaag tagaggctgc 960agtgagctat gattgcagca ctgcactgaa gccggggcaa cagaacaaga tccaaaaaaa 1020agggaggggt gaggggcaga gccaggattt gtttccaggc tgttgttacc taggtccgac 1080tcctggctcc cagagcagcc tgtcctgcct gcctggaact ctgagcaggc tggagtcatg 1140gagtcgattc ccagaatccc agagtcaggg aggctggggg caggggcagg tcactggaca 1200aacagatcaa aggtgagacc agcgtagggc tgcagaccag gccaggccag ctggacgggc 1260acaccatgag gtaggtgggc gcccacagcc tccctgcagg gtgtggggtg ggagcacagg 1320cctgggccct caccgcccct gccctgccca taggctgctg accctcctgg gccttctgtg 1380tggctcggtg gccaccccct tgggcccgaa gtggcctgaa cctgtgttcg ggcgcctggc 1440atcccccggc tttccagggg agtatgccaa tgaccaggag cggcgctgga ccctgactgc 1500accccccggc taccgcctgc gcctctactt cacccacttc gacctggagc tctcccacct 1560ctgcgagtac gacttcgtca aggtgccgtc aggacgggag ggctggggtt tctcagggtc 1620ggggggtccc caaggagtag ccagggttca gggacacctg ggagcagggg ccaggcttgg 1680ccaggaggga gatcaggcct gggtcttgcc ttcactccct c gtgacacctg cccacagc 1740tgagctcggg ggccaaggtg ctggccacgc tgtgcgggca ggagagcaca gacacggagc 1800gggcccctgg caaggacact ttctactcgc tgggctccag cctggacatt accttccgct 1860ccgactactc caacgagaag ccgttcacgg ggttcgaggc cttctatgca gccgagggtg 1920agccaagagg ggtcctgcaa catctcagtc tgcgcagctg gctgtggggg taactctgtc 1980ttaggccagg cagccctgcc ttcagtttcc ccacctttcc cagggcaggg gagaggcctc 2040tggcctgaca tcatccacaa tgcaaagacc aaaacagccg tgacctccat tcacatgggc 2100tgagtgccaa ctctgagcca gggatctgag gacagcatcg cctcaagtga cgcagggact 2160ggccgggcgc agcagctcac gcctgtaatt ccagcacttt gggaggccga ggctggctga 2220tcatttgagg tcaggagttc aaggccagcc agggcaacac ggtgaaactc tatctccact 2280aaaactacaa aaattagctg ggcgtggtgg tgcgcacctg gaatcccagc tactagggag 2340gctgaggcag gagaattgct tgaacctgcg aggtggaggc tgcagtgaac agagattgca 2400ccactacact ccagcctggg cgacagagct agactccgtc tcaaaaaaca aaaaacaaaa 2460acgacgcagg ggccgagggc cccatttaca gctgacaaagcgctgccagg atgtttgatt tcagatccca gtccctgcagggcaagtggc tcaatttctc tgctccttag gaagctgctgcccgccccct gggtttg att gactcccctc attagctgggaaactcccac tggtttaaca gaggtgatgt ttgcatctttttgcagcgac cctaggcctg taaggtgatt ggcccggcacgacgaggcct cctctgaggt ccactctgag gtcatggatcggatcccgcc tctttccctc ctgacggcct gcctggccctcgagtgccag gtggccccgg gagaggcgcc cacctgcgacgggcggtttc tactgctcct gccgcgcagg ctacgtcctgctcagccctg tgctccggcc aggtcttcac ccagaggtctatacccacgg ccgtatccca aactctccag ttgcacttacgttcagtgtc attctggact ttgtggagtc cttcgatgtggtgtccctac gactttctca agattcaaac agacagagaagaagacattg ccccacagga ttgaaacaaa aagcaacacgagatgaatca ggagaccaca caggctggaa gatccactaccccttatccg atggcgccac ctaatggcca cgtttcacctgaaagacagc ttctccatct tttgcgagac tggctatgagcctgaaatcc tttactgcag tttgtcagaa agatggatctgtgcagcatt gttgactgtg gccctcctga tgatctaccccacaggtcct ggagtgacca cctacaaagc tgtgattcagctacacaatg aaagtgaatg atggtaaata tgtgtgtgagctccaaagga gaaaaatcac tcccagtctg tgagcctgttaacaggaggg cgtatatatg gagggcaaaa ggcaaaacctcctgatatta ggtggaacca cagcagcagg tgcacttttaagctgctcat gccgtctatg agcaaaaaca tgatgcatcccaccctgaaa agactatcac ctcatta tac acaagcctggtgaaggttat actcatgatg ctggctttga caatgacatacaaagttgta atcaatagca acatcacgcc tatttgtctgctttatgagg acagatgaca ttggaactgc atctggatggtcttgctaga aatctaatgt atgtcgacat accgattgtttgcatatgaa aagccaccct atccaagggg aagtgtaactcttagaaagt gggggcaagg acagctgcag aggtgacagcagatagtgaa acagagaggt ggtttgtggg aggaatagtgtggggaagca ggtcagtatg gagtctacac aaaagttattgaacataatt agtgattttt aacttgcgtg tctgcagtcaatgcctgtga agaccttggc agcgacgtgg ctcgagaagctggcagttgt tgctccaccc aaaaaaacag actccaggtgcttgccagtt taattccagc cttacccatt gactcaagggcagtcatctt tgcccaccca gt.gtaatgtc actgctcaaaaagccagtct cttttcatac tggctgttgg catttctgtatgtttttaaa cttgttctta ttgaaaaaaa aaaaaaaaaa

tggggccctgagaccaactgcaagggttcatgacctcgggctcccagcgccagtcccgcatcctgggagggcctctcccccaccactgcccaccgtaacaggggagctcaagcatcagccgagacacaccgaacatggccgtgaccatcaacgagcacaggtgcaagccacttctgcaagtgggaccggcagtggccgagtacagctgtggctgatggattgtggactatggtgattttctatgacaactgccctggacatctgaagctggcactgattaccaagaaaagggattaacccgaccatcaaagctaacatgcggaggggcactcctggggttaactatattcaggattcttcattcatcattaggctgctgtgacataaaccttacatttcaaactgcctgt

ccagcgggagtgtgacctctgcgctgtagcccggacactgtgctgggagcccctagacagagtccaggctcagacattgaacaaccacctagcgcacctggcagccctgatggaggagggctgaaaccctcattctgtggcctttgtcaccgcacgcttgaatacatcctgtcacttgcccaatgcccgctggagtacataagagacctttctggacgagcagcccgcaccttggcaagtgggtcctaacttcgaatgggtttttatacaaattgaataaaagctgaatcaaaggggtttaatgtactgctttgtgctggtggtgtttctccatgaattgcctggatcgaatttttagaaactgtggacacatttctccaacgagagtgattaccttaaaccatgctctt

<210><211><212><213><220><221> <222> <4 00>

50

2090DNA

Murine MASP-2 CDS

(2090)

CDS (33) 50

ggcgctggac tgcagagcta tggtggcaca cc atg agg

Met arg1

ggt ctg ctg tgg agt ttg gtg gcc aca ctt ctgGly Leu Leu Trp Ser Leu Val Ala Thr Leu Leu

10 15

gaa cct gta ttc ggg cgg ctg gtg tcc cct ggcGlu Pro Val Phe Gly Arg Read Le Val Ser Pro Gly

25

30

gct gac cat caa gat cga tcc tgg aca ctg actAla Asp His Gln Asp Arg Ser Trp Thr Leu Thr

45

cta ctcLeu Leu

ggt tcaGly Ser20ttc ccaPhe Pro35

gca cccAla Pro

10

50

cgc ctg cgc ctc tac ttc acc cac tt gac ctg gaa ctc

until ttc ctgIle Phe Leu5

aag tgg cctLys Trp Pro

gag aag tatGlu Lys Tyr

cct ggc tacPro Gly Tyr55

tet tac cgc

25202580264027002760282028802940300030603120318032403300336034203480354036003660372037803840390039604020408041404404240454046204680474048004860492049.

53

101

149

197

24521

Arg Leu Arg Leu Tyr Phe Thr His Phe Asp Leu Glu Leu To Be Tyr Arg

60 65 70

tgc gag tat gac tt gtc aag ttg age tca ggg acc aag gtg ctg gcc 293

Cys Glu Tyr Asp Phe Val Lys Leu Be Ser Gly Thr Lys Val Leu Ala

75 80 85

aca ctg tgt ggg cag gag agt aca gac act gag cag gea cct ggc aat 341

Thr Read Cys Gly Gln Glu Be Thr Asp Thr Glu Gln Wing Pro Gly Asn

90 95 100

gac acc ttc tac tca ctg ggt ccc age cta aag gtc acc ttc cac tcc 389

Asp Thr Phe Tyr Be Read Gly Pro Be Read Lys Val Thr Phe His Be

105 110 115

gac tac tcc aag gag aag ccg ttc aa ggg ttt gag gcc ttc tat gea 437

Asp Tyr Ser Asn Glu Lys Pro Phe Thr Gly Phe Glu Wing Phe Tyr Wing120 125 130 135

gcg gag gat gtg gat gaa tgc aga gtg tet ctg gga gac tca gtc cct 485

Glu Asp Val Wing Asp Glu Cys Arg Val Val Ser Leu Gly Asp Val Val Ser

140 145 150

tgt gac cat tat tgc cac aac tac ttg ggc ggc tac tat tgc tcc tgc 533

Cys Asp His Tyr Cys His Asn Tyr Read Gly Gly Tyr Tyr Cys Ser Cys

155 160 165

aga gcg ggc tac att ctc cac cag aac aag cac acg tgc tca gcc ctt 581

Arg Wing Gly Tyr Ile Leu His Gln Asn Lys His Thr Cys Being Ala Leu

170 175 180

tgt tca ggc cag gtg ttc aca gga aga tet ggg tat ctc agt age cct 629

Cys Be Gly Gln Val Phe Thr Gly Arg Be Gly Tyr Read Be Ser Pro

185 190 195

gag tac ccg cag cca tac ccc aag ctc tcc age tgc time tac to 677

Glu Tyr Pro Gln Pro Tyr Pro Lys Read Be Cys Thr Tyr Be Ile200 205 210 215

cgc ctg gag gac ggc ttc agt gtc until ctg gac ttc gtg gag tcc ttc 725

Arg Read Le Glu Asp Gly Phe Ser Val Ile Read Le Glu Asp Phe Val Ser Phe

220 225 230

gat gtg gag acg cac cct gaa gcc cag tgc ccc tat gac tcc ctc aag 773

Asp Val Glu Thr His Pro Glu Wing Gln Cys Pro Tyr Asp Ser Leu Lys

235 240 245

att caa aca gac aag ggg gaa cac ggc cca ttt tgt ggg aag acg ctg 821

Ile Gln Thr Asp Lys Gly Glu His Gly Phe Cys Gly Lys Thr Leu

250 255 260

cct ccc agg att gaa act gac age cac aag gtg acc till acc ttt gcc 869

Pro Pro Arg Ile Glu Thr Asp Be His Lys Val Thr Ile Thr Phe Wing

265 270 275

act gac gag tcg ggg aac cac acg ggc tgg aag ata cac tac aca age 917

Thr Asp Glu Ser Gly Asn His Thr Gly Trp Lys Ile His Tyr Thr Ser280 285 290 295

aca gea cgg ccc tgc cct gat cca acg gcg cca cct aat ggc age att 965

Thr Wing Pro Pro Cys Pro Asp Pro Thr Wing Pro Pro Asn Gly Ser Ile

300 305 310

tca cct gtg caa gcc acg tat gtc ctg aag gac agg ttt tet gtc ttc 1013

Ser Pro Val Gln Wing Thr Tyr Val Leu Lys Asp Arg Phe Ser Val Phe

315 320 325

tgc aag aca ggc ttc gag ct ctg caa ggt tet gtc ccc ctg aaa tca 1061

Cys Lys Thr Gly Phe Glu Leu Leu Gln Gly Ser Val Pro Leu Lys Ser

330 335 340

ttc act gct gtc tgt cag aaa gat gga tet tgg gac cgg ccg atg cca 1109

Phe Thr Wing Val Cys Gln Lys Asp Gly Ser Trp Asp Arg Pro Met Pro

345 350 355

gag tgc age att att gat tgt ggc cct ccc gat gac cta ccc aat ggc 1157

Glu Cys Ser Ile Ile Asp Cys Gly Pro Pro Asp Asp Leu Pro Asn Gly360 365 370 375

cat gtg gac tat till aca ggc cct caa gtg act acc tac aaa gct gtg 1205

His Val Asp Tyr Ile Thr Gly Pro Gln Val Thr Thr Tyr Lys Wing Val380 385 390

att cag tac age tgt gaa gag act ttc tac aca atg age age aat ggt 1253Ile Gln Tyr

aaa tatLys Tyr

aaa ctcLys Leu425ata ggaIle Gly440

cct tggPro Trp

ctt ataLeu Ile

aaa agaLys Arg

aggArg

catHis520attIle

ctcLeu505gaaGlu

aaaLys

tgc ctaCys Leu

gga actGly Thr

aac ctaAsn Leu585acc gtgThr Val600

ctc tgtLeu Cys

agt gggSer Gly

gtg ggaVal Gly

cag tatGln Tyr665aac ataAsn Ile680 <210> <211> <212> <213> <400> Met Arg1

gtgVal410

CCC

Pro

ggaGly

caaGln

catHis

atgMet490tcaSer

ggcGly

ctcLeu

ccgPro

gtgVal570atgMet

tatTyr

gctAla

ggg

Gly

ggaGly650

ggg

Gly

ataIle

Ser Cys395

tgt gagCys Glu

ccg gttPro Val

cgc ataArg Ile

gtc ttgVal Leu4 60gac aatAsp Asn475

gca gcgAla Wing

cct catPro His

tac actTyr Thr

Glu Glu Thr

gct gat ggaAla Asp Gly415

tgt gag cctCys Glu Pro

430gtt gga gggVal Gly Gly445

ttg ctg ggtLeu Leu Gly

tgg gtc ctaTrp Val Leu

aagLys

cgaArg555gctAla

aacAsn540aaaLys

ggcGly

tccSer

tacTyr

cacHis525aaaLys

tccSer

actThr510ggtGly

ctgLeu495caaGln

gctAla

gtc acaVal Thr

gaa gct gcaGlu Ala Ala

ttt gtgPhe Val

gaa aagGlu Lys

ggcGly

gcaAla635ataIle

ttaLeu620ttaLeu

gttVal

tggTrp

gacAsp

ctcLeu605gagGlu

ggg ttaGly Leu575ata ccaIle Pro590

tat ccaTyr Pro

act ggtThr Gly

gtc tacVal Tyr

agt aatSer Asn

gtg ttt ctaVal Phe Leu

tcc tgg ggtSer Trp Gly655

aca aaa gtcThr Lys Val

670ttc taaPhe685

Phe400ttcPhe

gttVal

cagGln

caaGln

acaThr480aacAsn

gccAla

ggtGly

See you

tccSer560accThr

attIle

ggaGly

ggcGly

gatAsp640tccSer

See you

Tyr Thr Met

tgg acgTrp Thr

tgt gggCys Gly

cct gcaPro Ala450act acaThr Thr465

gcc gctAla Wing

until cgaIle Arg

tgg cccTrp Pro

tttPhe

aacAsn545ttaLeu

gacAsp530ggaGly

atgMet

cag aagGln Lys

gct gacAla Asp

gta agaVal Arg610aag gacLys Asp625

aat gagAsn Glu

att aatIle Asn

aac tatAsn Tyr

ageSer

ctgLeu435aagLys

gcaAla

catHis

atgMet

gagGlu515aatAsn

ageSer

agaArg

ggg

Gly

cacHis595gtaVal

ageSer

acaThr

tgtCys

attIle675

Being Ser Asn Gly405

tcc aaa gga gaaSer Lys Gly Glu420

tcc aca cac actSer Thr His Thr

cct ggtPro Gly

gca gcaAla Wing

gct gtaAla Val485ggc ateGly Ile500

gaa ateGlu Ile

gacAsp

ggtGly470tatTyr

tttPhe455gcaAla

gagGlu

ctc aaaLeu Lys

ttt ataPhe Ile

gatAsp

See you

acaThr

cttLeu580caaGln

ataIle

atgMet

gacAsp565cttLeu

gcaAla

cctPro550ttcPhe

ttgLeu535gttVal

actThr

gct agaAla Arg

aaa tgt accLys Cys Thr

age gct aacSer Ala Asn

tgc aga ggtCys Arg Gly630

cag cga tggGln Arg Trp645

ggg gcg gcaGly Wing Ala660

ccc tgg aatPro Trp Asn

atgMet615gacAsp

tttPhe

ggcGly

gagGlu

51

685

PRT

Murine51 MASP-2 CDS

Leu Leu Ile Phe Leu Gly5

1301

1349

1397

1445

1493

1541

1589

1637

1685

1733

1781

1829

1877

1925

1973

2021

2069

2090

Read

Leu Trp Ser Leu Val Wing Thr10 15Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val Ser

20 25 30

Pro Gly Phe Pro Glu Lys Tyr Asp Wing His Gln Asp Arg Be Trp Thr

35 40 45

Read Thr Ala Pro Pro Gly Tyr Arg Read Le Arg Read Le Tyr Phe Thr His Phe

50 55 60

Asp Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu Ser65 70 75 80

Be Gly Thr Lys Val Leu Wing Thr Read Cys Gly Gln Glu Be Thr Asp

85 90 95

Thr Glu Gln Wing Pro Gly Asn Asp Thr Phe Tyr Be Read Gly Pro Ser

100 105 110

Read Lys Val Thr Phe His Be Asp Tyr Be Asn Glu Lys Pro Phe Thr

115 120 125

Gly Phe Glu Wing Phe Tyr Wing Glu Wing Asp Val Asp Glu Cys Arg Val

130 135 140

Serly Gly Asp Serly Val Pro Cys Asp His Tyr Cys His Asn Tyr Leu145 150 155 160

Gly Gly Tyr Tyr Cys Be Cys Arg Wing Gly Tyr Ile Read His Gln Asn

165 170 175

Lys His Thr Cys Be Wing Read Cys Be Gly Gln Val Phe Thr Gly Arg

180 185 190

Being Gly Tyr Leu Being Being Pro Glu Tyr Pro Gln Pro Tyr Pro Lys Leu

195 200 205

Be Ser Cys Thr Tyr Ser Ile Arg Leu Glu Asp Gly Phe Ser Val Ile

210 215 220

Read Asp Phe Val Glu Be Phe Asp Val Glu Thr His Pro Glu Wing Gln225 230 235 240

Cys Pro Tyr Asp Being Read Lys Ile Gln Thr Asp Lys Gly Glu His Gly

245 250 255

Pro Phe Cys Gly Lys Thr Leu Pro Arg Ile Glu Thr Asp Be His

260 265 270

Lys Val Thr Ile Thr Phe Ala Thr Asp Glu Ser Gly Asn His Thr Gly

275 280 285

Trys Lys Ile His Tyr Thr Be Thr Arg Wing Pro Cys Pro Asp Pro Thr

290 295 300

Pro Wing Pro Asn Gly Ser Ile Ser Pro Val Gln Wing Thr Tyr Val Leu305 310 315 320

Lys Asp Arg Phe Be Val Phe Cys Lys Thr Gly Phe Glu Read Leu Gln

325 330 335

Gly Ser Val Pro Read Lys Be Phe Thr Wing Val Cys Gln Lys Asp Gly

340 345 350

Be Trp Asp Arg Pro Met Pro Glu Cys Be Ile Ile Asp Cys Gly Pro

355 360 365

Pro Asp Asp Read Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro Gln

370 375 380

Val Thr Thr Tyr Lys Wing Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe385 390 395 400

Tyr Thr Met Being Asn Gly Lys Tyr Val Cys Glu Wing Asp Gly Phe

405 410 415

Trp Thr Be Ser Lys Gly Glu Lys Leu Pro Pro Val Cys Glu Pro Val

420 425 430

Cys Gly Reads Being Thr His Thr Ile Gly Arg Ile Val Gly Gly Gln

435 440 445

Pro Wing Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly Gln

450 455 460

Thr Thr Wing Wing Wing Gly Wing Leu Ile His Asp Asn Trp Val Leu Thr465 470 475 480

Wing Wing His Wing Val Tyr Glu Lys Arg Met Wing Wing Ser Ser Leu Asn

485 490 495

Ile Arg Met Gly Ile Leu Lys Arg Leu Be Pro His Tyr Thr Gln Wing

500 505 510

Trp Pro Glu Glu Ile Phe Ile His Glu Gly Tyr Thr His Gly Wing Gly515 520 525

Phe Asp Asn Asp Ile Wing Leu Ile Lys Leu Lys Asn Lys Val Thr Ile

530 535 540

Asn Gly Ser Ile Met Pro Val Cys Leu Pro Arg Lys Glu Wing Wing Ser545 550 555 560

Leu Met Arg Thr Asp Phe Thr Gly Thr Val Wing Gly Trp Gly Leu Thr

565 570 575

Gln Lys Gly Leu Leu Arg Wing Arg Asn Leu Met Phe Val Asp Ile Pro Ile

580 585 590

Asp Wing His Gln Lys Cys Thr Thr

595 600 605

Val Arg Val Ser Wing Asn Met Leu Cys Wing Gly Leu Glu Thr Gly Gly

610 615 620

Lys Asp Be Cys Arg Gly Asp Be Gly Gly Wing Leu Val Phe Leu Asp625 630 635 640

Asn Glu Thr Gln Arg Trp Phe Gly Gly Ile Val Ser Trp Gly Ser

645 650 655

Ile Asn Cys Gly Wing Gly Wing Gly Gln Tyr Gly Val Tyr Thr Lys Val Ile

660 665 670

Asn Tyr Ile Pro Trp Asn Glu Asn Ile Ile Ser Asn Phe675 680 685

<210> 52 <211> 670 <212> PRT

<213> Mature murine MASP-2 protein <400> 52

Thr Read Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val1 5 10 15

Being Pro Gly Phe Pro Glu Lys Tyr Asp Wing His Gln Asp Arg Being Trp

20 25 30

Thr Leu Thr Wing Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His

35 40 45

Phe Asp Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu

50 55 60

Be Ser Gly Thr Lys Val Leu Wing Thr Read Cys Gly Gln Glu Ser Thr65 70 75 80

Asp Thr Glu Gln Pro Wing Gly Asn Asp Thr Phe Tyr Being Read Gly Pro

85 90 95

Ser Liu Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe

100 105 110

Thr Gly Phe Glu Wing Phe Tyr Wing Glu Wing Asp Val Asp Glu Cys Arg

115 120 125

Val Ser Read Gly Asp Ser Val Pro Cys Asp His Tyr

130 135 140

Read Gly Gly Tyr Tyr Cys Be Cys Arg Wing Gly Tyr Ile Read His Gln145 150 155 160

Asn Lys His Thr Cys Be Wing Read Cys Be Gly Gln Val Phe Thr Gly

165 170 175

Arg Be Gly Tyr Read Be Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys

180 185 190

Read Be Ser Cys Thr Tyr Be Ile Arg Read Glu Asp Gly Phe Ser Val

195 200 205

Ile Read Asp Phe Val Glu Be Phe Asp Val Glu Thr His Pro Glu Wing

210 215 220

Gln Cys Pro Tyr Asp Being Read Lys Ile Gln Thr Asp Lys Gly Glu His225 230 235 240

Gly Pro Phe Cys Gly Lys Thr Read Pro Pro Arg Ile Glu Thr Asp Ser

245 250 255

His Lys Val Thr Ile Thr Phe Ala Thr Asp Glu Ser Gly Asn His Thr

260 265 270

Gly Trp Lys Ile His Tyr Thr Be Thr Wing Arg Pro Cys Pro Asp Pro

275 280 285

Thr Wing Pro Pro Asn Gly Ser Ile Be Pro Val Gln Wing Thr Tyr Val290 295 300 Leu Lys Asp Arg Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu305 310 315 320Gln Gly Ser Val Pro Read Lys Ser Phe Thr Wing Val Cys Gln Lys Asp 325 330 335 Gly Be Trp Asp Arg Pro Met Pro Glu Cys Be Ile Ile Asp Cys Gly 340 345 350 Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro 355 360 365 Gln Val Thr Thr Val Ile Gln Tyr Be Cys Glu Glu Thr 370 375 380 Phe Tyr Thr Met Be Ser Asn Gly Lys Tyr Val Cys Glu Wing Asp Gly385 390 395 400Phe Trp Thr Be Ser Lys Gly Glu Lys Leu Pro Val Cys Glu Pro 405 410 415 Val Cys Gly Leu Be Thr His Thr Ile Gly Gly Arg Ile Val Gly Gly 420 425 430 Gln Pro Wing Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly 435 440 445 Gln Thr Thr Wing Wing Gly Wing Leu Ile His Asp Asn Trp Val Leu 450 455 460 Thr Wing His Wing Val Wing Tyr Glu Lys Ar g Met Wing Ala Ser Be Leu465 470 475 480Asn Ile Arg Met Gly Ile Leu Lys Arg Leu Be Pro His Tyr Thr Gln 485 490 495 Trp Pro Wing Glu Ile Phe Ile His Glu Gly Tyr Thr His Gly Wing 500 505 510 Gly Phe Asp Asn Asp Ile Wing Leu Ile Lys Leu Lys Asn Lys Val Thr 515 520 525 Ile Asn Gly Ser Ile Met Pro Val Cys Leu Pro Arg Lys Gla Wing 530 535 540 Ser Leu Met Arg Thr Asp Phe Thr Gly Thr Val Gly Trp Gly Leu545 550 555 560Thr Gln Lys Gly Leu Leu Wing Arg Asn Leu Met Phe Val Asp Ile Pro 565 570 575 Ile Wing Asp His Gln Lys Cys Thr Thr Val Tyr Cys Wing Gly Leu Glu Thr Gly 595 600 605 Gly Lys Asp To Be Cys Arg Gly Asp To Be Gly Gly Wing Alu Leu Val Phe Leu 610 615 620 Asp Asn Glu Thr Gln Arg Cys Gly Wing Gly Wing Gln Tyr Gly Val Tyr Thr Lys Val 645 650 655 Ile Asn Tyr Ile Pro Trp Asn Glu Asn

660 665 670

<210> 53 <211> 2091 <212> DNA

<213> MASP-2 Mouse CDS <220>

<221> CDS

<222> (10) .. (2067)

<400> 53

tggcacaca atg agg cta ctg up to gtc ctg ggt ctg ctt tgg agt ttg gtg 51

Met Arg Leu Leu Ile Val Leu Gly Leu Leu Trp Ser Leu Val1 5 10

gcc aca ctt ttg ggc tcc aag tgg cct gag cct gta ttc ggg cgc ctg 99

Wing Thr Read Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu15 20 25 30

gtg tcc ctg gcc ttc cca gag aag tat ggc aac cat cag gat cga tcc 147

Val Ser Leu Phe Pro Glu Wing Lys Tyr Gly Asn His Gln Asp Arg Sertgg acgTrp Thr

cac ttcHis Phe

ttg accLeu Thr80aca gatThr Asp95

ccc agePro Ser

ttc acaPhe Thr

aga acaArg Thr

tac ctgTyr Leu160cag aacGln Asn175

ggg aggGly Arg

aaa ctcLys Leu

until accIle Thr

gcc cagAla Gln240tac ggcTyr Gly255

age aacSer Asn

aca ggcThr Gly

cca acgPro Thr

gtc ctgVal Leu320ctg caaLeu Gln335

gat ggaAsp Gly

ggc cctGly Pro

35

ctg act gea ccc

Read Thr Ala Pro50

aac ctg gaa ctc

Asn Leu Glu Leu65

tca ggg acc aag

Be Gly Thr Lys

act gag cgg geaThr Glu Arg Ala100

cta aag gtc accLeu Lys Val Thr. 115

gga ttt gag gccGly Phe Glu Ala130

tcc ctg gga gacSer Leu Gly Asp145

ggc ggc tac tacly Gly Tyr Tyr

aag cat acc tgcLys His Thr Cys180

tet ggc ttt ctcSer Gly Phe Leu195

tcc age tgc gccSer Ser Cys Ala210

ctg gac ttc gtgLeu Asp Phe Val225

tgc ccc tac gacCys Pro Tyr Asp

ccg ttt tgt gggPro Phe Cys Gly260

aag gtg acc attLys Val Thr Ile275

tgg aag ata cacTrp Lys Ile His290

gcg cca cct aatAla Pro Pro Asn305

aag gac age tttLys Asp Ser Phe

gt tet gtc cccGly Ser Val Pro340

tet tgg gac cggSer Trp Asp Arg355

ccc gat gac ctaPro Asp Asp Leu

40

cct ggc ttc cgcPro Gly Phe Arg55

tet tac cgc tgcSer Tyr Arg Cys70

gtg cta gcc acgVal Read Ala Thr85

cct ggc aat gacPro Gly Asn Asp

tcc cac tcc gacPhe His Ser Asp120

ttc tat gea gcgPhe Tyr Ala Ala135

tca gtc cct tgtSer Val Pro Cys150

tgc tcc tgc cgaCys Ser Cys Arg165

tca gcc ctt tgtSer Ala Leu Cys

agt age cct gagSer Ser Pro Glu200

tac aac to cgcTyr Asn Ile Arg215

gag tcc ttt gatGlu Ser Phe Asp230

tcc ctc aag attSer Leu Lys Ile245

aag acg cct cccLys Thr Leu Pro

acc ttt acc accThr Phe Thr Thr280

tac aca age acaTyr Thr Ser Thr295

ggt cac att tcaGly His Ile Ser310

tet gtc ttc tgcSer Val Phe Cys325

ctg aag tca ttcLeu Lys Ser Phe

ccg lac cca gagPro Ile Pro Glu360

ccc aat ggc cacPro Asn Gly His

ctg cgc ctc tacLeu Arg Leu Tyr60

gag tat gac ttt

Glu Tyr Asp Phe75

ctg tgt ggg cagLeu Cys Gly Gln90

acc ttc tac tca

Thr Phe Tyr Ser105

tac tcc aat gag

Tyr Ser Asn Glu

gag gat gtg gat

Glu Asp Val Asp140

gac cat tat tgc

Asp His Tyr Cys155

gtg ggc tac att

Val Gly Tyr Ile170

tca ggc cag gtg

Ser Gly Gln Val185

tac cca cag cca

Tyr Pro Gln Pro

ctg gag gaa ggcLeu Glu Glu Gly220

gtg gag atg cacVal Glu Met His235

caa aca gac aagGln Thr Asp Lys250

ccg agg att gaaPro Arg Ile Glu265

gac gag tca gggAsp Glu Ser Gly

gea cag ccc tgcAla Gln Pro Cys300

cct gtg caa gcc · Pro Val Gln Ala315

aag act ggc ttcLys Thr Gly Phe330

act gct gtc tgtThr Val Wing Cys345

tgc age att attCys Ser Ile Ile

gtg gac tat ateVal Asp Tyr Ile

45

ttc acc 195

Phe thr

gtc aag 243

Val lys

gag agt 2 91

Glu Ser

ctg ggt 339

Read Gly110

aag cca 387

Lys Pro

125

gaa tgc 435

Glu Cys

cac aac 483

His asn

ctg cac 531

Read his

ttc act 579

Phe Thr190

tac ccc 627

Tyr pro

205

ttc agt 675

Phe Ser

cct gaa 723

Pro glu

agg gaa 771

Arg glu

act gac 819

Thr Asp270

aac cac 867

Asn his

285

cct gat 915

Pro Asp

acg tat 963

Thr tyr

gag ctt 1011

Glu Leu

cag aaa 1059

Gln Lys350

gac tgt 1107

Asp Cys

365

aca ggc 1155Thr Gly370 375 380

cct gaa gtg acc acc tac aaa gct gtg att cag tac age tgt gaa gag 1203

Pro Glu Val Thr Thr Tyr Lys Wing Val Ile Gln Tyr Ser Cys Glu Glu

385 390 395

act ttc tac aca atg age age aat ggt aaa tat gtg tgt gag gct gat 1251

Thr Phe Tyr Thr Met Be Asn Gly Lys Tyr Val Cys Glu Wing Asp

400 405 410

gga ttc tgg acg age tcc aaa gga gaa aaa tcc ctc ccg gtt tgc aag 1299

Gly Phe Trp Thr Be Lys Gly Glu Lys Be Le Pro Val Cys Lys415 420 425 430

cct gtc tgt gga ctg tcc aca cac act tca gga ggc cgt ata att gga 1347

Pro Val Cys Gly Read Be Thr His Thr Be Gly Gly Arg Ile Ile Gly

435 440 445

gga cag cct gea aag cct ggt gac ttt cct tgg caa gtc ttg tta ctg 1395Gly Gln Pro Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu

450 455 460

ggt gaa act aca gea gea ggt gct ctt ata cat gac gac tgg gtc cta 1443

Gly Glu Thr Thr Wing Gly Wing Leu Ile His Asp Asp Trp Val Leu

465 470 475

aca gcg gct cat gct gta tat ggg aaa aca gag gcg atg tcc tcc ctg 14 91

Thr Wing Ala His Wing Val Tyr Gly Lys Thr Glu Ala Met Be Ser Leu

480 485 490

gac till cgc atg ggc until ctc aaa agg ctc tcc ctc att tac act caa 1539

Asp Ile Arg Met Gly Ile Leu Lys Arg Leu Be Leu Ile Tyr Thr Gln495 500 505 510

gcc tgg cca gag gct gtc ttt until cat gaa ggc tac act cac gga gct 1587

Trp Pro Glu Wing Val Phe Ile Wing His Glu Gly Tyr Thr His Gly Wing

515 520 525

ggt ttt gac aat gat ata gea ctg att aaa ctc aag aaa gtc aca 1635

Gly Phe Asp Asn Asp Ile Wing Leu Ile Lys Leu Lys Asn Lys Val Thr

530 535 540

up to aac aga aac up tog ccg att tgt cta cca aga aaa gaa gct gea 1683

Ile Asn Arg Asn Ile Met Pro Ile Cys Read Pro Arg Lys Glu Wing Wing

545 550 555

tcc tta atg aaa aca gac ttc gt gg act gtg gct ggc tgg ggg tta 1731

Get Met Le Lys Thr Asp Phe Val Gly Thr Val Gly Wing Trp Gly Leu

560 565 570

acc cag aag ggg ttt ctt gct aga aac cta atg ttt gtg gac ata cca 1779

Thr Gln Lys Gly Phe Leu Arg Wing Asn Leu Met Phe Val Asp Ile Pro575 580 585 590

att gtt gac cac caa aaa tgt gct act gcg tat aca aag cag ccc tac 1827

Ile Val Asp His Gln Lys Cys Wing Thr Wing Tyr

595 600 605

cca gga gea aaa gtg act gtt aac atg ctc tgt gct ggc cta gac cgc 1875

Pro Gly Wing Lys Val Thr Val Asn Met Leu Cys Wing Gly Leu Asp Arg

610 615 620

ggt ggc aag gac age tgc aga ggt gac age gga ggg gea tta gtg ttt 1923

Gly Gly Lys Asp Being Cys Arg Gly Asp Being Gly Gly Wing Read Val Phe

625 630 635

cta gac aat gaa cag cg aga tgg ttt gtg gga gga ata gtt tcc tgg 1971

Read Asp Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp

640 645 650

ggt tet att aac tgt ggg ggg tca gaa cag tat ggg gtc tac acg aaa 2019

Gly Ser Ile Asn Cys Gly Gly Gly Glu Tyr Gly Val Tyr Thr Lys655 660 665 670

gtc acg aac tat att ccc tgg att gag aac ata aat aat aat ttc taa 2067

Val Thr Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Asn Ashe Phe675 680 685

tttgcaaaaa aaaaaaaaaa aaaa 2091

<210> 54 <211> 685 <212> PRT

<213> MASP-2 Mouse CDS <4 00> 54

Met Arg Leu Leu Ile Val Leu Gly1 5 Leu Leu Gly Ser Lys Trp Pro Glu 20 Leu Wing Phe Pro Glu Lys Tyr 35 40Leu Thr Wing Pro Gly Phe Arg 50 55 Asn Leu Glu Leu Ser Tyr Arg Cys65 70 Ser Gly Thr Lys Val Leu Wing Thr 85 Thr Glu Arg Wing Pro Gly Asn Asp 100 Leu Lys Val Thr Phe His Wing Ser Asp 115 120Gly Phe Glu Wing Phe Tyr Wing Wing 130 135 Ser Leu Gly Asp Being Val Pro Cys145 150 Gly Gly Tyr Tyr Cys Be Cys Arg 165 Lys His Thr Cys Be Ala Leu Cys 180 Be Gly Phe Leu Be Ser Pro Glu 195 200Ser Be Cys Ala Tyr Asn Ile Arg 210 215 Leu Asp Phe Val Glu Ser Phe Asp225 230 Cys Pro Tyr Asp Ser Leu Lys Ile 245 Pro Phe Cys Gly Lys Thr Leu Pro 260 Lys Val Thr Ile Thr Phe Thr 275 280Trp Lys Ile His Tyr Thr Be Thr 290 295 Pro Pro Wing Asn Gly His Ile Ser305 310 Lys Asp Be Phe Ser Val Phe Cys 325 Gly Be Val Pro Leu Lys Ser Phe 340 Ser Trp Asp Arg Ile Pro Glu 355 360Pro Asp Asp Leu Pro Asn Gly His 370 375 Val Thr Thr Tyr Lys Wing Val Ile385 390 Tyr Thr Met Being Ser Asn Gly Lys 405 Trp Thr Being Ser Lys Gly Glu Lys 420 Cys Gly Leu Being Thr His Thr 435 440Pro Lys Pro Wing Gly Asp Phe Pro 450 455 Thr Thr Wing Gly Wing Leu Wing Ile465 470 His Wing Val Tyr Gly Lys Thr 485 Wing

Leu Leu Trp Ser Leu Val Wing Thr 10 15 Pro Val Phe Gly Arg Leu Val Ser25 30 Asn His Gln Asp Arg Ser Trp Thr 45 Leu Arg Leu Tyr Phe Thr His Phe 60 Glu Tyr Asp Phe Val Lys Leu Thr 75 80Leu Cys Gly Gln Glu Be Thr Asp 90 95 Thr Phe Tyr Be Leu Gly Pro Ser105 110 Tyr Be Asn Glu Lys Pro Phe Thr 125 Glu Asp Val Asp Glu Cys Arg Thr 140 Asp His Tyr Cys His Asn Tyr Leu 155 160Val Gly Tyr Ile Leu His' Gln Asn 170 175 Ser Gly Gln Val Phe Thr Gly Arg 185 190 Tyr Pro Gln Pro Tyr Pro Lys Leu 205 Leu Glu Glu Gly Phe Ser Ile Thr 220 Val Glu Met His Pro Glu Wing 235 240Gln Thr Asp Lys Arg Glu Tyr Gly 250 255 Pro Arg Ile Glu Thr Asp Be Asn265 270 Asp Glu Be Gly Asn His Thr Gly 285 Wing Gln Pro Cys Pro Asp Pro Thr 300 Pro Val Gln Wing Thr Tyr Val Leu 315 320Lys Thr Gly Phe Glu Leu Read Gln 330 335 Thr Wing Val Cys Gln Lys Asp Gly345 350 Cys Ser Ile Ile Asp Cys Gly Pro 365 Val Asp Pro Glu 380 Gln Tyr Be Cys Glu Glu Thr Phe 395 400Tyr Val Cys Glu Wing Asp Gly Phe 410 415 Ser Leu Pro Val Cys Lys Pro Val425 430 Gly Gly Arg Ile Ile â € “Gly Gly GlnTrp Gln Val Leu Read Leu Gly Glu 460 His Asp Asp Trp Val Leu Thr Ala 475 480Glu Ala Met Ser Be Leu Asp Ile 490 495Arg Met Gly Ile Leu Lys Arg Leu Ser Leu Ile Tyr Thr Gln Ala Trp

500 505 510

Pro Glu Wing Val Phe Ile His Glu Gly Tyr Thr His Gly Wing Gly Phe

515 520 525

Asp Asn Asp Ile Wing Leu Ile Lys Leu Lys Asn Lys Val Thr Ile Asn

530 535 540

Arg Asn Ile Met Pro Ile Cys Leu Pro Arg Lys Glu Wing Wing Ser Leu545 550 555 560

Met Lys Thr Asp Phe Val Gly Thr Val Gly Wing Trly Gly Leu Thr Gln

565 570 575

Lys Gly Phe Leu Arg Wing Asn Leu Met Phe Val Asp Ile Pro Ile Val

580 585 590

Asp His Gln Lys Cys Wing Thr Thr Wing

595 600 605

Alys Lys Val Thr Val Asn Met Leu Cys Ala Gly Leu Asp Arg Gly Gly

610 615 620

Lys Asp Be Cys Arg Gly Asp Be Gly Gly Wing Leu Val Phe Leu Asp625 630 635 640

Asn Glu Thr Gln Arg Trp Phe Gly Gly Ile Val Ser Trp Gly Ser

645 650 655

Ile Asn Cys Gly Gly Be Glu Gln Tyr Gly Val Tyr Thr

660 665 670

Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Asn Ashe Phe675 680 685

<210> 55 <211> 670 <212> PRT

<213> Mature murine MASP-2 protein <400> 55

Thr Read Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val1 5 10 15

Being Read Ala Phe Pro Glu Lys Tyr Gly Asn His Gln Asp Arg Being Trp

20 25 30

Thr Leu Thr Wing Pro Pro Gly Phe Arg Leu Arg Leu Tyr Phe Thr His

35 40 45

Phe Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu

50 55 60

Thr Be Gly Thr Lys Val Leu Wing Thr Read Cys Gly Gln Glu Be Thr65 70 75 80

Asp Thr Glu Arg Wing Pro Gly Asn Asp Thr Phe Tyr Be Leu Gly Pro

85, 90 95

Ser Liu Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe

100 105 110

Thr Gly Phe Glu Wing Phe Tyr Wing Glu Wing Asp Val Asp Glu Cys Arg

115 120 125

Thr Be Read Gly Asp Be Val Pro Cys Asp His Tyr

130 135 140

Read Gly Gly Tyr Tyr Cys Be Cys Arg Val Gly Tyr Ile Read His Gln145 150 155 160

Asn Lys His Thr Cys Be Wing Read Cys Be Gly Gln Val Phe Thr Gly

165 170 175

Arg Be Gly Phe Read Be Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys

180 185 190

Read Ser Be Cys Wing Tyr Asn Ile Arg Read Glu Glu Gly Phe Ser Ile

195 200 205

Thr Read Asp Phe Val Glu Be Phe Asp Val Glu Met His Pro Glu Wing

210 215 220

Gln Cys Pro Tyr Asp Being Read Lys Ile Gln Thr Asp Lys Arg Glu Tyr225 230 235 240

Gly Pro Phe Cys Gly Lys Thr Read Pro Pro Arg Ile Glu Thr Asp Ser

245 250 255

Asn Lys Val Thr Ile Thr Thr Phe Thr Asp Glu Ser Gly Asn His Thr260 265 270Gly Trp Lys Ile His Tyr Thr Thr Wing Gln Pro Cys Pro Asp Pro 275 280 285 Thr Wing Pro Pro Asn Gly His Ile Ser Pro Val Gln Wing Thr Tyr Val 290 295 300 Leu Lys Asp Be Phe Be Val Phe Cys Lys Thr Gly Phe Glu Leu Leu305 310 315 320Gln Gly Be Val Pro Leu Lys Be Phe Thr Wing Val Cys Gln Lys Asp 325 330 335 Gly Ser Trp Asp Arg Pro Ile Pro Glu Cys Ser Ile Ile Asp Cys Gly 340 345 350 Pro Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro 355 360 365 Glu Val Thr Thr Tyr Lys Wing Val Ile Gln Tyr Ser Cys Glu Thr 370 375 380 Phe Tyr Thr Met Being Asn Gly Lys Tyr Val Cys Glu Wing Asp Gly385 390 395 400Phe Trp Thr Being Ser Lys Gly Glu Lys Being Le Pro Val Cys Lys Pro 405 410 415 Val Cys Gly Leu Being Thr His Thr Being Gly Gly Arg Ile Ile Gly Gly 420 425 430 Gln Pro Wing Lys Pro Gly Asp Phe Pro Trp Gln Val Le u Leu Leu Gly 435 440 445 Glu Thr Thr Wing Gly Wing Leu Ile His Asp Asp Trp Val Leu Thr 450 455 4 60 Wing His His Wing Val Tyr Gly Lys Thr Glu Wing Met Being Ser Leu Asp465 470 475 480Ile Arg Met Gly Ile Leu Lys Arg Leu Be Leu Ile Tyr Thr Gln Wing 485 490 495 Trp Pro Glu Wing Val Phe Ile His Glu Gly Tyr Thr His Gly Wing Gly 500 505 510 Phe Asp Asn Asp Ile Wing Leu Ile Lys Leu Lys Asn Lys Val Thr Ile 515 520 525 Asn Arg Asn Ile Met Pro Ile Cys Leu Pro Arg Lys Glu Wing Ala Ser 530 535 540 Leu Met Lys Thr Asp Phe Val Gly Thr Val Gly Trp Gly Leu Thr545 550 555 560Gln Lys Gly Phe Read Wing Arg Asn Leu Met Phe Val Asp Ile Pro Ile 565 570 575 Val Asp His Gln Lys Cys Wing Thr Thr Wing Tyr Thr Lys Gln Pro Tyr 580 585 590 Gly Wing Lys Val Thr Val Asn Met Leu Cys Wing Gly Leu Asp Arg Gly 595 600 605 Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Wing Leu Val Phe Leu 610 615 620 Asp Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly625 630 635 640Ser Ile Asn Cys Gly Gly Be Glu Tyr Val Tyr Val Tyr Thr Lys Val 645 650 655 Thr Asn Tyr Ile Pro Trp Ile Glu Asn Ile Asn Asn Phe 660 665 670 <210> 56 <211> 28

<212> DNA

<213> Artificial sequence <220>

<223> Homo Sapien MASP-2 PCR Primer

<400> 56

atgaggctgc tgaccctcct gggccttc

<210> 57

<211> 23

<212> DNA

<213> Artificial sequence <220> <223> Homo Sapien MASP-2 PCR primer <400> 57

gtgcccctcc tgcgtcacct ctg <210> 58 <211> 23 <212> DNA

<213> Artificial sequence <220>

<223> Homo Sapien MASP-2 PCR Primer <400> 58

cagaggtgac gcaggagggg cac <210> 59 <211> 27 <212> DNA

<213> Artificial sequence <220>

<223> Homo Sapien MASP-2 PCR Primer

<400> 59

ttaaaatcac taattatgtt ctcgatc <210> 60

<211> 22

<212> DNA

<213> Artificial sequence <220>

<223> Murine MASP-2 PCR Primer

<400> 60

atgaggctac tcatcttcct gg

<210> 61

<211> 23

<212> DNA

<213> Artificial sequence <220>

<223> Murine MASP-2 PCR Primer

<400> 61

ctgcagaggt gacgcagggg ggg

<210> 62

<211> 23

<212> DNA

<213> Artificial sequence <220>

<223> Murine MASP-2 PCR Primer

<400> 62

ccccccctgc gtcacctctg cag

<210> 63

<211> 29

<212> DNA

<213> Artificial sequence <220>

<223> Murine MASP-2 PCR Primer

<400> 63

ttagaaatta cttattatgt tctcaatcc

<210> 64

<211> 29

<212> DNA

<213> Artificial sequence <220>

<223> Mouse MASP-2 Oligonucleotide

<400> 64

gaggtgacgc aggaggggca ttagtgttt

<210> 65

<211> 37

<212> DNA

<213> Artificial sequence <220>

<223> Mouse MASP-2 oligonucleotide <400> 65

ctagaaacac taatgcccct cctgcgtcac ctctgca

Claims (58)

A method for inhibiting MASP-2 dependent complement activation in a subject in need thereof, comprising comprising administering to the subject an amount of a MASP-2 inhibitor effective to inhibit MASP-2 dependent complement activation. Method according to claim 1, characterized in that the MASP-2 inhibiting agent specifically binds to a polypeptide comprising SEQ ID NO: 6. A method according to claim 1, characterized in that the MASP-2 inhibitory agent specifically binds to a polypeptide comprising SEQ ID NO: 6 with a 10-fold greater affinity of the same than to a polypeptide. different antigen in the complement system. Method according to claim 2, characterized in that the MASP-2 inhibitory agent binds to the polypeptide at a site within amino acid residues 1 to 176 of SEQ ID NO: 6. A method according to claim 1, characterized in that the MASP-2 inhibiting agent is an antibody or fragment thereof that specifically binds a portion of SEQ ID NO: 6. Method according to claim 5, characterized in that the antibody or fragment thereof is monoclonal. Method according to claim 5, characterized in that the antibody or fragment thereof is polyclonal. A method according to claim 5, characterized in that the antibody or fragment thereof is a recombinant antibody. Method according to claim 5, characterized in that the antibody has reduced effector function. A method according to claim 5, characterized in that the antibody is a chimeric, humanized or human antibody. A method according to claim 5, characterized in that the antibody is produced in a MASP-2 deficient transgenic animal. A method according to claim 1, characterized in that the MASP-2 inhibitory agent is a peptide derived from a polypeptide selected from the group consisting of human MASP-2, human MBLh that inhibits MASP-2, human H-phytoline. inhibits MASP-2, human M-phytoline that inhibits MASP-2, human L-phytoline that inhibits MASP-2 and human C4 that inhibits MASP-2. A method according to claim 1, characterized in that the MASP-2 inhibiting agent is a non-peptide agent that specifically binds to a polypeptide comprising SEQ ID NO: 6. A method according to claim 13, characterized in that the MASP-2 inhibiting agent binds to the polypeptide at a site within amino acid residues 1 to 176 of SEQ ID NO: 6. A method for inhibiting MASP-2 dependent complement activation in an individual in need thereof, comprising administering to the subject an amount of MASP-2 inhibitor effective to selectively inhibit MASP-2 dependent complement activation. 2 without substantially inhibiting Clq-dependent complement activation. A method according to claim 15, characterized in that the MASP-2 inhibiting agent specifically binds to a polypeptide comprising SEQ ID NO: 6. A method according to claim 16, characterized in that the MASP-2 inhibiting agent binds to the polypeptide at a site within amino acid residues 1 to 176 of SEQ ID NO: 6. A method according to claim 15, characterized in that the MASP-2 inhibiting agent is an antibody or fragment test that specifically binds a portion of SEQ ID NO: 6. A method according to claim 18, characterized in that the antibody or fragment thereof is monoclonal. The method of claim 18, wherein the antibody is a chimeric, humanized or human antibody. The method of claim 18, wherein the antibody is produced in a MASP-2 deficient transgenic animal. The method according to claim 15, characterized in that the MASP-2 inhibiting agent is a peptide derived from a polypeptide selected from the group consisting of human MASP-2, human MBLh that inhibits MASP-2, human H-phytoline. inhibits MASP-2, human L-ficoline that inhibits MASP-2 and human C4 that inhibits MASP-2. The method of claim 15, characterized in that the MASP-2 inhibiting agent is a non-peptide agent that specifically binds to a polypeptide comprising SEQ ID NO: 6. Composition for inhibiting MASP-2 dependent complement activation, characterized in that it comprises a therapeutically effective amount of a MASP-2 inhibiting agent and a pharmaceutically acceptable carrier. 25. A method for manufacturing a medicament for use in inhibiting the effects of MASP-2-dependent complement activation on living individuals in need thereof, which comprises combining a therapeutically effective amount of a MASP-2 inhibitor into a pharmaceutical carrier. . A method for treating an individual suffering from a MASP-2 dependent complement-mediated condition, said condition being a vascular condition, characterized in that it comprises administering an amount of an effective MASP-2 inhibiting agent to inhibit complement-dependent activation. of MASP-2. A method according to claim 26, characterized in that the vascular condition is selected from the group consisting of a cardiovascular condition, a cerebrovascular condition, a peripheral vascular condition (eg, musculoskeletal), a renal vascular condition, a mesenteric / enteric vascular condition. , revascularization for transplants and / or replantations, vasculitis, Henoch-Schonlein purpura nephritis, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis, vasculitis of immune complex, Takayasu's disease, cardiomyopathy, diabetic disease, ), poisonous gas plunger (VGE) and restenosis following probing, rotational atherectomy, and percutaneous transluminal coronary angioplasty (PTCA). A method for treating an individual suffering from a MASP-2-dependent complement-mediated condition associated with an ischemia-reperfusion injury, characterized in that administering an amount of an effective MASP-2 inhibitor to inhibit complement-dependent activation. of MASP-2. A method according to claim 28, characterized in that the ischemia-reperfusion injury is associated with aortic aneurysm repair, cardiopulmonary bypass, vascular reanastomosis, organ transplantation and / or extremity / finger reimplantation, stroke. , myocardial infarction, and hemodynamic resuscitation following shock and / or surgical procedures. A method for treating and / or preventing atherosclerosis in an individual in need thereof, which comprises administering an amount of an effective MASP-2 inhibiting agent to inhibit MASP-2 dependent complement activation. 31. Method for treating an individual suffering from MASP-2-dependent complement-mediated condition associated with a common inflammatory gastrointestinal disorder, which comprises administering an amount of an effective MASP-2-inhibiting agent to inhibit MASP-dependent complement activation. -2. A method according to claim 31, characterized in that the inflammatory gastrointestinal disorder is selected from the group consisting of pancreatitis, Crohn's disease, ulcerative colitis, irritable bowel syndrome and diverticulitis. 33. A method for treating an individual suffering from a MASP-2 dependent complement-mediated condition, said condition being a pulmonary condition, characterized in that it comprises administering an amount of an effective MASP-2 inhibiting agent to inhibit complement-dependent activation. of MASP-2. The method according to claim 33, characterized in that the pulmonary condition is selected from the group consisting of acute respiratory distress syndrome, transfusion-related acute lung injury, acute ischemia / reperfusion lung injury, chronic obstructive pulmonary disease, asthma, granulomatosis. Wegener's disease, antiglomerular-based demembrane disease (Goodpasture's disease), meconium aspiration syndrome, bronchiolitis obliterative syndrome, pulmonary idiopathic fibrosis, acute pulmonary injury secondary to burn, cardiogenic pulmonary edema, transfusion-related respiratory depression and emphysema. 35. A method for inhibiting MASP-2 dependent complement activation in an individual who has undergone, is undergoing or will undergo an extracorporeal reperfusion procedure, characterized in that it comprises administering an amount of a MASP-2 inhibiting agent effective to inhibit activation. of MASP-2-dependent complement. A method according to claim 35, characterized in that the extracorporeal reperfusion procedure is selected from the group consisting of hemodialysis, plasmapheresis, leukopheresis, extracorporeal demembrane oxygenator (ECMO), precipitation of heparin-induced extracorporeal membrane oxygenation (HELP). ) and cardiopulmonary bypass (CPB). 37. A method for treating an individual suffering from a MASP-2 dependent complement-mediated condition, said condition being a musculoskeletal condition, which comprises administering an amount of an effective MASP-2 inhibiting agent to inhibit complement-dependent activation. of MASP-2. 38. The method according to claim 37, characterized in that the musculoskeletal condition is selected from the group consisting of osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, gout, neuropathic arthropathy, spondyloarthropathy, crystaline arthropathy, muscular dystrophy, and lupus erythematosus. (SLE). 39. A method for treating an individual suffering from a MASP-2 dependent complement-mediated condition, said condition being a renal condition, characterized in that it comprises administering an amount of an effective MASP-2 inhibiting agent to inhibit complement-dependent activation. of MASP-2. 40. The method according to claim 39, characterized in that the renal condition is selected from the group consisting of mesangioproliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis (post-stromal glomerulonephritis), post-streptocellular glomerulonephritis, post-streptocellular glomerulonephritis of lupus, Henoch-Schonlein purpura nephritis and IgA nephropathy. 41. A method for treating an individual suffering from a MASP-2-dependent complement-mediated condition, said condition being a skin condition, which comprises administering an amount of an effective MASP-2 inhibiting agent to inhibit complement activation. dependent on MASP-2. Method according to claim 41, characterized in that the skin condition is selected from the group consisting of psoriasis, autoimmune bullous dermatoses, eosinophilic spongiosis, bullous pemphigoid, acquired bullous epidermolysis, thermal burn injury and burn injury. chemistry. 43. A method for inhibiting MASP-2 dependent complement activation in an individual who has undergone, is undergoing or will undergo an organ or tissue transplantation procedure, which comprises administering an amount of an effective MASP-2 inhibitor to inhibit MASP - 2 - dependent complement activation. A method according to claim 43, characterized in that the transplant procedure is selected from the group consisting of organ allograft, organ xenograft, organ graft and tissue. 45. Method for treating an individual suffering from a MASP-2-dependent complement-mediated condition associated with a common nervous system disorder or injury, which comprises administering an amount of an effective MASP-2 inhibiting agent to inhibit complement activation. of MASP dependent. A method according to claim 45, characterized in that the disorder or injury of the nervous system is selected from the group consisting of multiple sclerosis, myasthenia gravis, Huntington's disease, amyotrophic lateral sclerosis, Guillain Barre syndrome, reperfusion following stroke. degenerative discs, brain trauma, Parkinson's disease, Alzheimer's disease, Miller-Fisher syndrome, brain trauma and / or hemorrhage, demyelination and meningitis. 47. A method for treating an individual suffering from a MASP-2-dependent complement-mediated condition associated with a common blood disorder, which comprises administering an amount of an effective MASP-2 inhibiting agent to inhibit MASP-2-dependent complement activation. 2. The method according to claim 47, characterized in that the blood disorder is selected from the group consisting of septicemia, severe septicemia, septic shock, acute respiratory distress syndrome resulting from septicemia, systemic inflammatory response syndrome, hemorrhagic shock, hemolytic anemia. , autoimmune thrombotic thrombocytopenic purpuratrocytic and hemolytic uremic syndrome. 49. A method for treating an individual suffering from a MASP-2-dependent complement-mediated condition associated with a urogenital condition, which comprises administering an amount of an effective MASP-2 inhibitor to inhibit MASP-2-dependent complement activation. 2. A method according to claim 49, characterized in that the urogenital condition is selected from the group consisting of painful bladder disease, sensitive bladder disease, non-bacterial chronic cystitis, interstitial cystitis, infertility, placental dysfunction and abortion and preeclampsia. . 51. Method for treating an individual suffering from a MASP-2-dependent complement-mediated condition associated with diabetes in nonobese (Type 1 diabetes or insulin-dependent diabetes mellitus) and / or complications associated with Type 1 or Type 2 diabetes ( adulthood), characterized in that it comprises administering an amount of an effective MASP-2 inhibiting agent to inhibit MASP-2-dependent complement activation. The method of claim 51, characterized in that the complication associated with Type 1 or Type 2 diabetes is selected from the group consisting of angiopathy, neuropathy, and retinopathy. 53. A method for inhibiting MASP-2 dependent complement activation in a subject who has undergone, is undergoing or will undergo chemotherapy and / or radiation therapy, characterized in that it comprises administering an amount of an effective MASP-2 inhibiting agent. inhibit MASP-2-dependent complement activation. 54. A method of treating an individual suffering from a malignancy comprising administering a quantity of a MASP-2 inhibiting agent effective to inhibit MASP-2 dependent complement activation. 55. A method for treating an individual suffering from an endocrine disorder, which comprises administering a quantity of a MASP-2 inhibiting agent effective to inhibit MASP-2 dependent complement activation. A method according to claim 55, characterized in that the endocrine disorder is selected from the group consisting of Hashimoto's thyroiditis, stress, anxiety, hormonal disorders that involve the regulated release of prolactin, growth factor or other insulin-equivalent growth factor. and dapituitary adrenocorticotropin. 57. A method for treating an individual suffering from complement-mediated ophthalmic condition, which comprises administering an amount of an effective MASP-2 inhibiting agent to inhibit MASP-2-dependent complement activation. A method according to claim 57, characterized in that the ophthalmic condition is age-related macular degeneration.